Membrane layers for analyte sensors

ABSTRACT

Disclosed are devices for determining an analyte concentration (e.g., glucose). The devices comprise a sensor configured to generate a signal associated with a concentration of an analyte and a sensing membrane located over the sensor. The sensing membrane comprises a biointerface layer which interfaces with a biological fluid containing the analyte to be measured. The biointerface layer can comprises a biointerface polymer, wherein the biointerface polymer comprises polyurethane and/or polyurea segments and one or more zwitterionic repeating units. The sensing membrane can also comprise an enzyme layer, wherein the enzyme layer comprises an enzyme and a polymer comprising polyurethane and/or polyurea segments and one or more zwitterionic repeating units. The sensing membrane can also comprise a diffusion-resistance layer, which can comprise a base polymer having a lowest Tg of greater than −50 C.

INCORPORATION BY REFERENCE TO RELATED APPLICATIONS

Any and all priority claims identified in the Application Data Sheet, orany correction thereto, are hereby incorporated by reference under 37CFR 1.57. This application claims the benefit of U.S. ProvisionalApplication No. 62/273,155, filed Dec. 30, 2015; U.S. ProvisionalApplication No. 62/273,142, filed Dec. 30, 2015; and U.S. ProvisionalApplication No. 62/273,219, filed Dec. 30, 2015. Each of theaforementioned applications is incorporated by reference herein in itsentirety, and each is hereby expressly made a part of thisspecification.

FIELD

The subject matter disclosed herein relates to devices for measuring abiological analyte in a host and to components of such devices.

BACKGROUND

Electrochemical sensors are useful for determining the presence orconcentration of a biological analyte, such as blood glucose. Suchsensors are effective, for example, at monitoring glucose in diabeticpatients and lactate during critical care events. A variety ofintravascular, transcutaneous and implantable sensors have beendeveloped for continuously detecting and quantifying blood analytes,such as blood glucose levels.

A challenge associated with long-term use of continuous sensors andother medical devices is biomaterial-associated inflammation, which isthe inflammation caused by implantation of foreign materials into thebody. Biomaterial-associated inflammation is the result of a dynamicmicroenvironment around an implanted device, which includes the initialinjury, neutrophil and macrophage recruitment, foreign body giant cell(FBGC) response, neovascularization, fibroblast recruitment, anddownstream fibrosis. The formation of a barrier cell layer around theimplanted device can result in impaired tissue integration.

In the case of a continuous sensor, biomaterial-associated inflammationcan impair analyte transport from tissues to the sensor surface, whetherby creating a diffusion barrier or active consumption of analytes. TheFBGC response to implantable materials results in fibrous capsuleformation that impedes sensor function through the modulation of glucosediffusion through dense fibrotic “scar-tissue” layers. Otherconstituents of the biomaterial-associated inflammatory cascade, such asinflammatory cytokines and other small molecules, sometimes act asinterferents to sensor performance. These interferents can cause sensorinaccuracy due to the constant change in transport properties at thesensor/tissue interface. Aside from diffusion limitations, there existsan active cellular component of the biomaterial-associated inflammatorycascade, involving inflammatory and wound healing cells; these cellsthat can be activated by implanted materials can actively consumeglucose and produce H₂O₂, which can also lead to sensor inaccuracy.Reduction in biomaterial-associated inflammation is thus important increating long-term, stable, implantable sensors and devices. Thus whatare needed are methods and compositions that can reduce inaccuracies inan implanted sensor that can be caused by biomaterial-associatedinflammation. What are also needed are methods and compositions that canincrease the longevity of an implanted device. The methods andcompositions disclosed herein address these and other needs.

Also, in such analyte sensors there is a membrane layer or domain thatcontains an enzyme responsible for converting the analyte into agentthat can be registered as a measurable signal. For example, glucosesensors contain enzymes that convert glucose into hydroperoxide, whichis further converted into a sensor signal. So the performance ofenzymatic glucose sensors, like other sensors that rely on enzymaticconversions, can be affected by the amount of active enzyme incorporatedin the sensor's membrane layer.

It is often a challenge to have sufficient active enzyme incorporatedand maintained in the membrane to efficiently catalyze analyte reactions(e.g., glucose to hydrogen peroxide). Enzyme can leach out of themembrane in hydrated conditions. Leached enzyme can also result in asevere Foreign Body Response (FBR). These events change sensorsensitivity and degrade the resistance layer, which will finallydecrease the accuracy and longevity of the sensor. Furthermore, enzymedegradation can occur by many different mechanisms leading toirreversible or reversible inactivation of enzyme. Enzymes can besensitive to environment conditions including, for example, temperaturechanges, pH changes, and exposure to the reactive chemicals includingcrosslinkers often employed in immobilization the enzyme such asglutaraldehyde and carbodiimide, as well as bi-products from the redoxreaction such as hydrogen peroxide and gluconic acid and endogenousbi-products. Enzyme degradation severely limits the functional life ofanalyte sensors in vivo and can result in gradual sensor sensitivitydecline and early onset of sensor end-of-life.

The incorporation and immobilization of enzymes into various carriers orbinders including polymers, sol gel, particles, and mixtures thereof tocreate an enzyme layer has been tried to prevent the leaching of enzymesin analyte sensors. These layers can, however, swell and degrade inaqueous environments and suffer from poor adhesion to adjacent layers inthe membrane system. As a result, these by-products can also leach outof the membrane and also contribute to the FBR and affect sensorsensitivity and accuracy. Poor adhesion can further result in reducedmechanical stability and delamination of the membrane layers in vivo.

There is thus a desire for engineered enzyme layers in which enzymes areimmobilized in the membrane via strong molecular level interactionsbetween enzymes and base polymeric materials. Such layers can reduce (orprevent) leaching of enzymes, which will lessen the FBR, and improve thelongevity, sensitivity, and accuracy of the sensor. What are also neededare enzyme layers engineered with physiochemical stability and catalyticperformance stability in aqueous environment and have good adhesion toother layers in the sensor's membrane system. The compositions, methods,and devices disclosed herein address these and other needs.

Further, in such analyte sensors there is a membrane layer or domainthat is primarily responsible for limiting the diffusion of the analyteto the sensor. The function of this so-called diffusion-resistance layercan be important when the analyte of interest is present in the patientin amounts that can exceed the sensor's sensitivity. For example, thereexists a molar excess of glucose relative to the amount of oxygen in thebody. So for every free oxygen molecule in extracellular fluid, thereare typically more than 100 glucose molecules present (see Updike etal., Diabetes Care 5:207-21 (1982)). However, an immobilizedenzyme-based glucose sensor employing oxygen as co-reactant ispreferably supplied with oxygen in non-rate-limiting excess in order forthe sensor to respond linearly to changes in glucose concentration,while not responding to changes in oxygen concentration. Specifically,when a glucose-monitoring reaction is oxygen limited, linearity is notachieved above minimal concentrations of glucose. Without asemipermeable membrane situated over the enzyme domain to control theflux of glucose and oxygen, a linear response to glucose levels can beobtained only for glucose concentrations of up to about 40 mg/dL.However, in a clinical setting, a linear response to glucose levels isdesirable up to at least about 400 mg/dL.

Some diffusion-resistance layers suffer from changes in permeability toanalyte due to changes in microstructure, phase separation, degradation,and interferants from external molecules. These changes can affectsensor sensitivity and therefore result in changes in sensor output forthe same analyte concentration over time—a phenomenon known as “drift.”This also confounds the development of robust algorithms due to the needfor sensor “drift” correction. There is thus a desire for an engineeredsemipermeable diffusion-resistance layer that contains polymers capableof providing a structurally stable matrix wherein an analyte permeablephase resides throughout, and electrochemical sensors equipped with suchmembrane. The compositions, methods, and devices disclosed hereinaddress these and other needs.

SUMMARY

In accordance with the purposes of the disclosed materials and methods,as embodied and broadly described herein, the disclosed subject matter,in one aspect, relates to compounds, compositions and methods of makingand using compounds and compositions, and devices containing compoundsand compositions.

In a first aspect, a device is provided for determining an analyteconcentration (e.g., glucose), the device comprising: a sensorconfigured to generate a signal associated with a concentration of ananalyte and a sensing membrane located over the sensor. The sensingmembrane comprises a biointerface layer which interfaces with abiological fluid containing the analyte to be measured. In devices ofthis aspect, the biointerface layer comprises a biointerface polymer,wherein the biointerface polymer comprises polyurethane and/or polyureasegments and one or more zwitterionic repeating units.

A biointerface layer increases sensor longevity and decrease sensorinaccuracy by inhibiting accumulation of cells, proteins, and otherbiological species on the outermost layers of the sensor. Earlyattenuation of these events in the biomaterial-associated inflammationcascade can lessen the overall severity of the response, and thereforethe sensor inaccuracy in vivo can be reduced.

In further embodiments of the disclosed devices the sensing membranefurther comprises an enzyme domain comprising an enzyme selected fromthe group consisting of glucose oxidase, glucose dehydrogenase,galactose oxidase, cholesterol oxidase, amino acid oxidase, alcoholoxidase, lactate oxidase, and uricase. In certain embodiments, theenzyme is glucose oxidase.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units comprise a betaine compound or derivative thereof. Inexamples of devices of this aspect, the one or more zwitterionicrepeating units comprise a betaine compound or precursor thereof.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units comprise at least one moiety selected from the groupconsisting of a carboxyl betaine, a sulfo betaine, a phosphor betaine,and derivatives thereof.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R², R³, and R⁴ are independently chosen from        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and wherein one or more of R¹, R², R³, R⁴, and Z are        substituted with a polymerization group.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³, are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³ are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In embodiments of devices of this aspect, wherein the polymerizationgroup is selected from alkene, alkyne, epoxide, lactone, amine,hydroxyl, isocyanate, carboxylic acid, anhydride, silane, halide,aldehyde, and carbodiimide.

In embodiments of devices of this aspect, the one or more zwitterionicrepeating units is at least about 1 wt. % based on the total weight ofthe polymer.

In embodiments of devices of this aspect, the polyurethane and/orpolyurea segments are from about 15 wt. % to about 75 wt. %, based onthe total weight of the polymer.

In embodiments of devices of this aspect, the biointerface polymerfurther comprises at least one segment selected from the groupconsisting of epoxides, polyolefins, polysiloxanes, polyamide,polystylene, polyacrylate, polyethers, polyesters, and polycarbonates.

In embodiments of devices of this aspect, the biointerface polymerfurther comprises a polyethylene oxide segment, which in some examplesis from about 5 wt. % to about 60 wt. %, based on the total weight ofthe biointerface polymer.

In embodiments of devices of this aspect, the biointerface polymer has amolecular weight of from about 10 kDa to about 500,000 kDa, apolydispersity index of from about 1.4 to about 3.5, and/or a contactangle of from about 20° to about 90°.

In a second aspect, a device is provided where the biointerface layerfurther comprises one or more zwitterions selected from the groupconsisting of cocamidopropyl betaine, oleamidopropyl betaine, octylsulfobetaine, caprylyl sulfobetaine, lauryl sulfobetaine, myristylsulfobetaine, palmityl sulfobetaine, stearyl sulfobetaine, betaine(trimethylglycine), octyl betaine, phosphatidylcholine, glycine betaine,poly(carboxybetaine), poly(sulfobetaine), and derivatives thereof.

In an embodiment of the second aspect, a device is provided where thebiointerface layer further comprises a pharmaceutical or bioactiveagent.

In a third aspect, a device is provided for determining an analyteconcentration, the device comprising: a sensor configured to generate asignal associated with a concentration of an analyte and a sensingmembrane located over the sensor. The sensing membrane comprises abiointerface layer which interfaces with a biological fluid containingthe analyte to be measured. In devices of this aspect, the biointerfacedomain comprises a biointerface polymer, wherein the biointerfacepolymer comprises a polymer chain having both hydrophilic andhydrophobic regions and wherein the hydrophilic regions comprise a linerpolymer chain having hydrophilic oligomers bound thereto and where thelinear polymer is grafted to the biointerface polymer.

In a fourth aspect, a device is provided for determining an analyteconcentration, the device comprising: a sensor configured to generate asignal associated with a concentration of an analyte and a sensingmembrane located over the sensor. The sensing membrane comprises abiointerface layer which interfaces with a biological fluid containingthe analyte to be measured. In devices of this aspect, the biointerfacelayer comprises a biointerface polymer, wherein the biointerface polymercomprises a fluorescent moiety covalently bonded to the biointerfacepolymer.

In a fifth aspect, a device is provided for determining an analyteconcentration, the device comprising: a sensor configured to generate asignal associated with a concentration of an analyte and a sensingmembrane located over the sensor. The sensing membrane comprises abiointerface layer which interfaces with a biological fluid containingthe analyte to be measured. In devices of this aspect, the biointerfacelayer comprises a base polymer and a surface modifying polymer, whereinthe surface modifying polymer comprises a polymer chain having bothhydrophilic and hydrophobic regions and wherein one or more zwitterioniccompounds; and wherein the base polymer is selected from silicone,epoxide, polyolefin, polystylene, polyoxymethylene, polysiloxane,polyether, polyacrylic, polymethacrylic, polyester, polycarbonate,polyamide, poly(ether ketone), poly(ether imide), polyurethane, andpolyurethane urea.

In a sixth aspect, a device is provided for determining an analyteconcentration (e.g., glucose), the device comprising: a sensorconfigured to generate a signal associated with a concentration of ananalyte and a sensing membrane located over the sensor. The sensingmembrane comprises an enzyme layer, wherein the enzyme layer comprisesan enzyme and a polymer comprising polyurethane and/or polyurea segmentsand one or more zwitterionic repeating units. The enzyme layer protectsthe enzyme and prevents it from leaching from the sensing membrane intoa host, without adversely affecting the enzyme's activity. The enzymelayer can be from 0.01 μm to about 250 μm thick.

In a seventh aspect, a device is provided for determining an analyteconcentration (e.g., glucose), the device comprising: a sensorconfigured to generate a signal associated with a concentration of ananalyte and a sensing membrane located over the sensor. The sensingmembrane comprises an enzyme layer, wherein the enzyme layer comprisesan enzyme and a polymer comprising polyurethane and/or polyurea segmentsand one or more zwitterionic repeating units. The enzyme layer protectsthe enzyme and prevents it from deactivating by dynamic changes in itsenvironment caused by endogenous and exogenous compounds, and otherstress factors including temperature and pH. The enzyme layer can befrom 0.01 μm to about 250 μm thick. In further examples of the devicesof the disclosed devices the enzyme can selected from the groupconsisting of glucose oxidase, glucose dehydrogenase, galactose oxidase,cholesterol oxidase, amino acid oxidase, alcohol oxidase, lactateoxidase, and uricase. In certain examples, the enzyme is glucoseoxidase.

In examples of devices of this aspect, the one or more zwitterionicrepeating units comprise a betaine compound or derivative thereof. Inexamples of devices of this aspect, the one or more zwitterionicrepeating units comprise a betaine compound or precursor thereof.

In examples of devices of this aspect, the one or more zwitterionicrepeating units comprise at least one moiety selected from the groupconsisting of a carboxyl betaine, a sulfo betaine, a phosphor betaine,and derivatives thereof.

In examples of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R², R³, and R⁴ are independently chosen from        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and wherein one or more of R¹, R², R³, R⁴, and Z are        substituted with a polymerization group.

In examples of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³, are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In examples of devices of this aspect, the one or more zwitterionicrepeating units are derived from a monomer selected from the groupconsisting of:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³ are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In examples of devices of this aspect, wherein the polymerization groupis selected from alkene, alkyne, epoxide, lactone, amine, hydroxyl,isocyanate, carboxylic acid, anhydride, silane, halide, aldehyde, andcarbodiimide.

In examples of devices of this aspect, the one or more zwitterionicrepeating units is at least about 1 wt. % based on the total weight ofthe polymer.

In examples of devices of this aspect, the polyurethane and/or polyureasegments are from about 15 wt. % to about 75 wt. %, based on the totalweight of the polymer.

In examples of devices of this aspect, the polymer in the enzyme layerfurther comprises at least one segment selected from the groupconsisting of epoxides, polyolefins, polysiloxanes, polyamide,polystylene, polyacrylate, polyethers, polyesters, and polycarbonates.

In examples of devices of this aspect, the polymer in the enzyme layerfurther comprises a polyethylene oxide segment, which in some examplesis from about 5 wt. % to about 60 wt. %, based on the total weight ofthe enzyme layer polymer.

In examples of devices of this aspect, the polymer in the enzyme layerhas a molecular weight of from about 10 kDa to about 500,000 kDa, apolydispersity index of from about 1.4 to about 3.5, and/or a contactangle of from about 10° to about 90°.

In a eight aspect, a device is provided where the enzyme layer furthercomprises a base polymer and enzyme stabilizing and/or immobilizingpolymer, wherein the enzyme stabilizing and/or immobilizing polymercomprises a polymer chain having both hydrophilic and hydrophobicregions and one or more zwitterionic repeating units; and wherein thebase polymer is selected from silicone, epoxide, polyolefin,polystylene, polyoxymethylene, polysiloxane, polyether, polyacrylic,polymethacrylic, polyester, polycarbonate, polyamide, poly(etherketone), poly(ether imide), polyurethane, and polyurethane urea.

In a ninth aspect, a device is provided where the enzyme layer furthercomprises an enzyme stabilizing reagent. In certain examples the enzymestabilizing reagent can be selected from the group consisting of one ormore zwitterions selected from the group consisting of cocamidopropylbetaine, oleamidopropyl betaine, octyl sulfobetaine, caprylylsulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmitylsulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octylbetaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine),poly(sulfobetaine), and derivatives thereof.

In a tenth aspect, devices are provided for determining an analyteconcentration (e.g., glucose), the devices comprising: a sensorconfigured to generate a signal associated with a concentration of ananalyte and a sensing membrane located over the sensor. In certainembodiments disclosed herein are devices for measurement of an analyteconcentration, the devices comprising a sensor configured to generate asignal associated with a concentration of an analyte; and a sensingmembrane located over the sensor, the sensing membrane comprising adiffusion-resistance layer comprising a base polymer having a lowestglass transition temperature as measured using ASTM D3418 of greaterthan −50° C. and an ultimate tensile strength as measured by ASTM D1708that is greater than 6000 psi. For example, the glass transitiontemperature of the base polymer can be greater than 0° C. and/or theultimate tensile strength of the base polymer can be greater than 8250psi.

In certain examples, the base polymer can be a segmented blockcopolymer. For example, the base polymer can comprise polyurethaneand/or polyurea segments and one or more polycarbonate or polyestersegments. In other examples, the base polymer can be a polyurethanecopolymer chosen from a polyether-urethane-urea, polycarbonate-urethane,polyether-urethane, polyester-urethane, and/or copolymers thereof, solong as the lowest glass transition temperature is greater than −50° C.In other examples, the base polymer comprises a polymer selected fromepoxies, polyolefins, polyoxymethylene, polyethers, polyacrylics,polymethacrylic, polyesters, polycarbonates, polyamide, poly(etherketone), poly(ether imide), and/or copolymers thereof so long as thelowest glass transition temperature is greater than −50° C. In stillother examples, the base polymer can be substantially free of silicone.In further examples, the diffusion-resistance layer further can comprisea hydrophilic polymer. For example, the hydrophilic polymer can beselected from polyvinyl alcohol, polyethylene glycol, polyacrylamide,polyacetate, polyethylene oxide, polyethyleneamine, polyvinylpyrrolidone(PVP), polyoxazoline (PDX), and copolymers and/or mixtures thereof. Inother examples, the hydrophilic polymer can be blended with the basepolymer or can be covalently bonded to the base polymer. In still otherexamples, the base polymer or hydrophilic polymer can comprise acrosslinker or several crosslinkers, where in the crosslinker comprise apolymer or oligomer selected from polyfunctional isocynate,polyfunctional aziridine, polyfunctional carbodiimide. In otherexamples, the diffusion-resistance layer can comprise a blend of apolycarbonate-urethane base polymer and polyvinylpyrrolidone. Thediffusion-resistance layer can be from 0.01 μm to about 250 μm thick.

In certain embodiments, disclosed are sensors that have a drift of lessthan or equal to 10% over 10 days. The sensor can comprise an electrodeand the device can be configured for continuous measurement of ananalyte concentration, such as glucose.

In other aspects, disclosed are devices for measurement of an analyteconcentration, the devices comprising: a sensor configured to generate asignal associated with a concentration of an analyte; and a sensingmembrane located over the sensor, the sensing membrane comprising adiffusion resistance layer, wherein the sensor has less than 10% driftin the signal over 10 days.

In still other aspects, disclosed are devices for measurement of ananalyte concentration, the devices comprising: a transcutaneous sensorconfigured to generate a signal associated with a concentration of ananalyte; and a sensing membrane located over the sensor, the sensingmembrane comprising a diffusion-resistance layer, wherein thediffusion-resistance layers comprises a polyurethane containing blockcopolymer and is substantially free of silicone.

In all of the devices disclosed herein, they can be configured forcontinuous measurement of an analyte concentration.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or can belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is a schematic view of a continuous analyte sensor systemattached to a host and communicating with other devices.

FIGS. 2A-2C are cross-sectional views of a sensor illustrating variousembodiments of the membrane system.

FIG. 3A is a side view schematic illustrating an in vivo portion of acontinuous analyte sensor, in one embodiment.

FIG. 3B is a perspective view schematic illustrating an in vivo portionof a continuous analyte sensor, in one embodiment.

FIG. 3C is a side view schematic illustrating an in vivo portion of acontinuous analyte sensor, in one embodiment.

FIG. 3D is a cross-sectional/side-view schematic illustrating an in vivoportion of a continuous analyte sensor, in one embodiment.

FIG. 4 is a schematic showing certain embodiments of a biointerfacepolymer.

FIG. 5 is a graph showing the T95 response (in seconds) of a continuoussensor without a biointerface layer (“no BL”) and one with abiointerface layer (“with BL”), as described herein. There is nosignificant difference in T95 response times between the sensors.

FIG. 6 is a graph showing the time lag from continuous glucose sensorswithout a biointerface layer (“RL”) and with biointerface layer SBL-3 orX-SBL-9 in a pig model. Polymers SBL-3 is from Table 1. Polymer X-SBL-9is from Table 1 but crosslinked.

FIG. 7 is a graph showing the calibration check performance ofcontinuous glucose sensors with and without a biointerface layer (SBL-8with 10% crosslinking). The biointerface layer did not significantlyaffect the bench performance of the sensor.

FIG. 8 is a group of photographs of sensors coated with three differentbiointerface layers. SBL-8 and SBL-10 are polymers from Table 1. SBL8/10is a 50/50 wt. % blend of polymers SBL-8 and SBL-10. As thehydrophilicity of the biointerface layer increases (as referenced by thepercentage of the polyethylene oxide in the polymer), the mechanicalstrength decreases. The decrease in mechanical strength is evident bythe small dark areas, which indicate compromises in the layer.

FIG. 9 is a graph of the hydrophobicity of various biointerface layersas measured by the amount of water uptake. SBL-3 is from Table 1.X-SBL-8 is polymer SBL-8 from Table 1 crosslinked. X-SBL-9 is polymerSBL-9 from Table 1 crosslinked. X-SBL-10 is polymer SBL-10 from Table 1crosslinked. X-CBL-3 is polymer SBL-3 from Table 1 where thesulfobetaines are replaced with carboxyl betaines and crosslinked.X-CBL-8 is polymer SBL-8 from Table 1 where the sulfobetaines arereplaced with carboxyl betaines and crosslinked. As the percentage ofhydrophilic segments (e.g., PEG) increases from SBL-3 to SBL-10, theamount of water uptake, and thus hydrophilicity, increases.

FIG. 10A is a graph of water absorption rate and tensile strength datafor crosslinked (X-SBL-8) and uncrosslinked (SBL-8) biointerfacepolymer. The data show that crosslinking results in faster waterabsorption and equilibration.

FIG. 10B is a graph of tensile strength data for crosslinked (X-SBL-8)and uncrosslinked (SBL-8) biointerface polymer. The data show thatcrosslinking increases the tensile strength.

FIG. 11 is a graph of crosslinking reaction kinetics as measured byFT-IR. The crosslinking reaction was done on polymer SBL-10 usingdifferent isocyanate-based crosslinkers.

FIG. 12 is a diagram illustrating possible mechanism for the antifoulingcharacteristics of a biointerface polymer layer.

FIG. 13 contains data for XPS studies on polymer SBL-3, SBL-10 dry, andSBL-10 soaked. The data show that there is no preferential migration ofbetaine to the surface of the biointerface layer when the sensor issoaked in water (supported by comparing the element S atom ratio ofSBL-10 coated dry sensor and SBL-10 coated sensor soaked). The XPS dataalso indicate sulfobetaine groups appear on the surface of sensor tipswith both SBL-3 and SBL-10 coated sensors (supported by observingelement S on the sensor tips). The increased amount of S atom detectedon the surface of BL-10 sample as compared to BL-3 sample is the resultof the increased loading of sulfo betaine in the polymer backbone.

FIG. 14 is a diagram illustrating a biointerface layer made of denselypacked hydrophilic brushes. The biointerface layer is grafted ontosurface functional groups in an adjacent layer (e.g., resistance layer).

FIG. 15 contains a chemical structure for a biointerface layer usinghydrophobic hyperbranched fluoropolymer (HBFP)-rich domains andhydrophilic PEG domains.

FIG. 16 is a pair of photographs showing a dry sensor coated with abiointerface layer and the same sensor after a 3-minute soak inphosphate buffer solution. The images show the biointerface layer swells(from 50 to 400%) after the soak, which indicates that the biointerfacelayer can be beneficial for excluding inflammatory cytokines from theimplantation site.

FIG. 17 is a diagram illustrating the use of additional functionality onthe biointerface layer. This functionality can be used to attach, e.g.,via click chemistry, antifouling reagents or proteins.

FIG. 18 is a graph showing the contact angle of various biointerfacelayers from Table 1. The variation of contact angle indicate that thewettability of the biointerface layer can be tuned by varying theamounts of hydrophobic and/or hydrophilic segments.

FIG. 19 is a graph showing the amount of protein (bovine serum albumin(BSA) or fibrinogen) absorption on different biointerface layers. Thereference layer (RL) does not contain a biointerface polymer.Biointerface layers where a betaine is within the main polymer chain arecompared with layers where the betaine is only at the ends of thepolymer chain.

FIG. 20 is a graph showing the protein adsorption via the QCM method onvarious sensors without biointerface polymers and those withbiointerface polymers. A silicone polycarbonate urethane layercorresponds to RL-1 and a silicone-free polycarbonate urethanecorresponds to RL-2. Biointerface layers SBL-1, SBL-3, and SBL-10 arefrom Table 1. As is seen when compared to sensors without a biointerfacelayer, the amount of protein adsorption is significantly less.

FIG. 21 is a graph of protein adsorption using an on-sensor micro-BCAmethod. The sensor without the biointerface layer (RL-1) hadsignificantly more protein than sensors coated with biointerface layersSBL-3 and SBL-10.

FIG. 22 is a graph of protein adsorption using a dye-labeled proteinadsorption screening method. Sensors with a layer lacking betaines (RL-7from Table 1) and control sensors without a biointerface layer (RL-1 andRL-2) showed significantly more protein adsorption than SBL-3,crosslinked versions of SBL-10, SBL-8, and SBL-9 (XSBL-10, XSBL-8, andXSBL-9, respectively), and crosslinked and uncrosslinked versions ofSBL-3 with carboxylbetains instead of sulfobetaines (XCBL-3 and CBL-3,respectively).

FIG. 23 is a graph showing the fibrous capsule thickness of a sensorwith (“BL”) and without (“No BL”) a biointerface layer. The biointerfacepolymer in the BL was SBL-3 from Table 1. The reduction in fibrouscapsule thickness when using a biointerface layers is significant.

FIG. 24 is a group of photographs showing stability of a sensor coatedwith a biointerface layer. The stable explant was coated with SBL-8 andthe unstable explant was coated with SBL-10. The dark areas indicatecompromises in the layer.

FIGS. 25A-D comprise a group of graphs showing the raw counts fromvarious sensors at 2 days and 14 days post implant. In the sensorwithout the biointerface layer (“No BL”) the spread of data points ismore pronounced at day 14 than at day 2, indicating a shift raw countsthat is due to biofouling. In contrast, the sensor with the biointerfacelayer (“with BL”) had very little spread of data points at 14 days; itwas similar to the spread at day 2, indicating that there was lessbiofouling.

FIG. 26A is a schematic view of a portion of one embodiment of aninterference domain that comprises a plurality of polycationic andpolyanionic layers.

FIG. 26B illustrates one embodiment of a layer-by-layer depositionmethod, which employs alternating adsorption of polycations andpolyanions to create a structure illustrated in FIG. 26A.

FIG. 27 is a graph of the 95% confidence interval for nominal noise forsensors coated with CBL-8 and SBL3.

FIG. 28 is a graph of ultimate tensile strength of SBL-3 and CBL-8, witha resistance layer as the control (RL) (i.e., a membrane in which therewas no biointerface layer and the resistance layer formed the outermostlayer).

FIG. 29 is a graph of tensile strain at maximum load of SBL-3 and CBL-8,with a resistance layer as the control (RL).

FIG. 30 is a graph of Young's modulus at 100% extension of SBL-3 andCBL-8, with a resistance layer as the control (RL).

FIG. 31 contains images of the skive regions of sensors with BSA-488incubated on various polymer surfaces after 1 hour.

FIG. 32 is a graph of normalized protein adsorption based on ImageJquantification of fluorescence intensity in skive region of sensor.

FIG. 33 is a schematic of a biointerface layer with Factor H covalentlylinked thereto through a linker.

FIG. 34 is a graph showing the % of active enzyme leached out over timeinto water from a 200 μm thick film of a control polymer blend with ahydrophilic polymer additive not containing betaine (P3) or from apolymer blend with hydrophilic polymer additive containing betaine inthe polymer backbone as disclosed herein.

FIG. 35 is a graph comparing various sensor metrics for glucose sensorsconstructed with an enzyme layer formed of the same polymer binder usedin (P3) but with 30 wt % of betaine containing polymer as enzymeimmobilization polymer additive vs. glucose sensors (P3) constructedwith an enzyme layer without the above-described 30 wt % betainecontaining polymer.

FIG. 36 is a graph showing the normalized elution of total proteinenzyme over time into water from a 200 μm thick film of a controlpolymer without betaines, a control polymer with small molecule betainesadded to the formulation, or a polymer with betaines in the polymerbackbone as disclosed herein.

FIG. 37 is a graph showing the water uptake over time for enzyme layerprepared from WB-7 and a control polymer, without betaines, (P3).

FIG. 38 is a graph showing the sensitivity of a sensor with a betainecontaining polymer in the enzyme layer and a sensor without such apolymer. The sensors coated with enzyme layers were treated at 70° C.and at 95% humidity. After this accelerated ageing treatment, theresistance layer was added and sensitivity was measured. The data show asteady dip in sensitivity for the sensor without the betaine containingpolymer, which is the result of enzyme deactivation due to thermalstress and/or high humidity.

FIG. 39 is a graph showing the accuracy of a sensor with a betainecontaining polymer in the enzyme layer and a sensor without such apolymer. The sensors were coated with enzyme layers and treated 70° C.and at 95% humidity. After this accelerated ageing treatment, theresistance layer was added sensor performance in the form of sensoraccuracy was measured and expressed as mean absolute relative difference(MARD), which is calculated from the average of absolute relativedifference of calculated value from least square linear fitting andactual value: Average of |(V_(cal)−V_(actual))/V_(actual)| for eachsteps of glucose concentration.

FIG. 40 is a graph showing results from an adhesion pull test.

FIG. 41 is a graph showing other results from the adhesion pull test.

FIG. 42 is a graph showing drift testing of sensors constructed fromsilicone containing diffusion resistance layer (RL) membrane (thin line)and silicone free diffusion resistance (RL) membrane (thick line).

FIG. 43 is a plot of differential scanning calorimetry heating scansperformed with TA Instrument Q2000 DSC using aluminum sample pans onresistance layer polyurethane polymer samples.

FIGS. 44A and 44B are a pair of graphs showing the tensile strength andpuncture resistance of silicone containing diffusion resistance (RL)membrane and silicone free diffusion resistance (RL) membrane. The datafor the tensile strength was obtained on membranes without blending witha hydrophilic polymer like PVP. The puncture resistance data wasobtained on wet membranes with blending with PVP.

FIG. 45 shows the results from an in vivo sensor stability test (Pigmodel).

FIG. 46 shows images captured by a scanning electron microcope ofsensors from the in vivo sensor stability test described in FIG. 45.

FIGS. 47A and 47B are a pair of graphs showing the sensor data in a pigmodel using sensors with a silicone containing diffusion resistance (RL)and sensors without silicone in the diffusion resistance RL.

FIG. 48 is a graph showing drift testing of sensors constructed from asilicone free diffusion resistance (RL) membrane soaked in water beforecuring. The control represents sensors constructed with a silocnecontaining RL with a standard humidity cure.

FIG. 49 is a schematic showing an embodiment of a blooming moiety and anetworking moiety.

FIG. 50 is a schematic showing an embodiment where the blooming andnetworking moiety is a siloxane.

FIG. 51 is a schematic showing an embodiment wherein the blooming moietyis tris(trimethylsilyl)siloxane and the networking moiety ismethacrylamide.

FIG. 52 is a schematic showing an embodiment wherein the blooming moietyis tris(trimethylsilyl)siloxane and the networking moiety is carboxylicacid.

DETAILED DESCRIPTION

The methods, compositions, and devices described herein can beunderstood more readily by reference to the following detaileddescription of specific aspects of the disclosed subject matter and theExamples and Figures included therein.

Before the methods, compositions, and devices are disclosed anddescribed, it is to be understood that the aspects described below arenot limited to specific synthetic methods or specific reagents, as suchcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “analyte” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to a substance or chemical constituent in abiological fluid (e.g., blood, interstitial fluid, cerebral spinalfluid, lymph fluid, urine, sweat, saliva, etc.) that can be analyzed.Analytes can include naturally occurring substances, artificialsubstances, metabolites, or reaction products. In some embodiments, theanalyte for measurement by the sensing regions, devices, and methods isglucose. However, other analytes are contemplated as well, including,but not limited to: acarboxyprothrombin; acylcarnitine; adeninephosphoribosyl transferase; adenosine deaminase; albumin; α-fetoprotein;amino acid profiles (arginine (Krebs cycle), histidine/urocanic acid,homocysteine, phenylalanine/tyrosine, tryptophan); andrenostenedione;antipyrine; arabinitol enantiomers; arginase; benzoylecgonine (cocaine);biotinidase; biopterin; c-reactive protein; carnitine; carnosinase; CD4;ceruloplasmin; chenodeoxycholic acid; chloroquine; cholesterol;cholinesterase; conjugated 1-β hydroxy-cholic acid; cortisol; creatinekinase; creatine kinase MM isoenzyme; cyclosporin A; d-penicillamine;de-ethylchloroquine; dehydroepiandrosterone sulfate; DNA (acetylatorpolymorphism, alcohol dehydrogenase, alpha 1-antitrypsin, cysticfibrosis, Duchenne/Becker muscular dystrophy, glucose-6-phosphatedehydrogenase, hemoglobin A, hemoglobin S, hemoglobin C, hemoglobin D,hemoglobin E, hemoglobin F, D-Punjab, β-thalassemia, hepatitis B virus,HCMV, HIV-1, HTLV-1, Leber hereditary optic neuropathy, MCAD, RNA, PKU,Plasmodium vivax, sexual differentiation, 21-deoxycortisol);desbutylhalofantrine; dihydropteridine reductase; diptheria/tetanusantitoxin; erythrocyte arginase; erythrocyte protoporphyrin; esterase D;fatty acids/acylglycines; free β-human chorionic gonadotropin; freeerythrocyte porphyrin; free thyroxine (FT4); free tri-iodothyronine(FT3); fumarylacetoacetase; galactose/gal-1-phosphate;galactose-1-phosphate uridyltransferase; gentamicin; glucose-6-phosphatedehydrogenase; glutathione; glutathione perioxidase; glycocholic acid;glycosylated hemoglobin; halofantrine; hemoglobin variants;hexosaminidase A; human erythrocyte carbonic anhydrase I;17-α-hydroxyprogesterone; hypoxanthine phosphoribosyl transferase;immunoreactive trypsin; lactate; lead; lipoproteins ((a), B/A-1, β);lysozyme; mefloquine; netilmicin; phenobarbitone; phenyloin;phytanic/pristanic acid; progesterone; prolactin; prolidase; purinenucleoside phosphorylase; quinine; reverse tri-iodothyronine (rT3);selenium; serum pancreatic lipase; sissomicin; somatomedin C; specificantibodies (adenovirus, anti-nuclear antibody, anti-zeta antibody,arbovirus, Aujeszky's disease virus, dengue virus, Dracunculusmedinensis, Echinococcus granulosus, Entamoeba histolytica, enterovirus,Giardia duodenalisa, Helicobacter pylori, hepatitis B virus, herpesvirus, HIV-1, IgE (atopic disease), influenza virus, Leishmaniadonovani, leptospira, measles/mumps/rubella, Mycobacterium leprae,Mycoplasma pneumoniae, Myoglobin, Onchocerca volvulus, parainfluenzavirus, Plasmodium falciparum, poliovirus, Pseudomonas aeruginosa,respiratory syncytial virus, rickettsia (scrub typhus), Schistosomamansoni, Toxoplasma gondii, Trepenoma pallidium, Trypanosomacruzi/rangeli, vesicular stomatis virus, Wuchereria bancrofti, yellowfever virus); specific antigens (hepatitis B virus, HIV-1);succinylacetone; sulfadoxine; theophylline; thyrotropin (TSH); thyroxine(T4); thyroxine-binding globulin; trace elements; transferrin;UDP-galactose-4-epimerase; urea; uroporphyrinogen I synthase; vitamin A;white blood cells; and zinc protoporphyrin. Salts, sugar, protein, fat,vitamins, and hormones naturally occurring in blood or interstitialfluids can also constitute analytes in certain embodiments. The analytecan be naturally present in the biological fluid or endogenous, forexample, a metabolic product, a hormone, an antigen, an antibody, andthe like. Alternatively, the analyte can be introduced into the body orexogenous, for example, a contrast agent for imaging, a radioisotope, achemical agent, a fluorocarbon-based synthetic blood, or a drug orpharmaceutical composition, including but not limited to: insulin;ethanol; cannabis (marijuana, tetrahydrocannabinol, hashish); inhalants(nitrous oxide, amyl nitrite, butyl nitrite, chlorohydrocarbons,hydrocarbons); cocaine (crack cocaine); stimulants (amphetamines,methamphetamines, Ritalin, Cylert, Preludin, Didrex, PreState, Voranil,Sandrex, Plegine); depressants (barbiturates, methaqualone,tranquilizers such as Valium, Librium, Miltown, Serax, Equanil,Tranxene); hallucinogens (phencyclidine, lysergic acid, mescaline,peyote, psilocybin); narcotics (heroin, codeine, morphine, opium,meperidine, Percocet, Percodan, Tussionex, Fentanyl, Darvon, Talwin,Lomotil); designer drugs (analogs of fentanyl, meperidine, amphetamines,methamphetamines, and phencyclidine, for example, Ecstasy); anabolicsteroids; and nicotine. The metabolic products of drugs andpharmaceutical compositions are also contemplated analytes. Analytessuch as neurochemicals and other chemicals generated within the body canalso be analyzed, such as, for example, ascorbic acid, uric acid,dopamine, noradrenaline, 3-methoxytyramine (3MT),3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA),5-hydroxytryptamine (5HT), and 5-hydroxyindoleacetic acid (FHIAA).

The term “baseline” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to the component of an analyte sensor signalthat is not related to the analyte concentration. In one example of aglucose sensor, the baseline is composed substantially of signalcontribution due to factors other than glucose (for example, interferingspecies, non-reaction-related hydrogen peroxide, or other electroactivespecies with an oxidation potential that overlaps with hydrogenperoxide). In some embodiments wherein a calibration is defined bysolving for the equation y=mx+b, the value of b represents the baselineof the signal.

The term “continuous (or continual) analyte sensing” as used herein is abroad term, and is to be given its ordinary and customary meaning to aperson of ordinary skill in the art (and is not to be limited to aspecial or customized meaning), and refers without limitation to theperiod in which monitoring of analyte concentration is continuously,continually, and or intermittently (but regularly) performed, forexample, about every 5 to 10 minutes.

The term “counts” as used herein is a broad term, and is to be given itsordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), andrefers without limitation to a unit of measurement of a digital signal.In one example, a raw data stream measured in counts is directly relatedto a voltage (for example, converted by an A/D converter), which isdirectly related to current from the working electrode. In anotherexample, counter electrode voltage measured in counts is directlyrelated to a voltage.

The term “dipole” or “dipolar compound” as used herein is a broad term,and is to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refer without limitation to compounds in whicha neutral molecule of the compound has a positive and negativeelectrical charge at different locations within the molecule. Thepositive and negative electrical charges within the molecule can be anynon-zero charges up to and including full unit charges.

The term “distal to” as used herein is a broad term, and is to be givenits ordinary and customary meaning to a person of ordinary skill in theart (and is not to be limited to a special or customized meaning), andrefers without limitation to the spatial relationship between variouselements in comparison to a particular point of reference. For example,some embodiments of a sensor include a membrane system having abiointerface domain and an enzyme domain. If the sensor is deemed to bethe point of reference and the biointerface domain is positioned fartherfrom the sensor than the enzyme domain, then the biointerface domain ismore distal to the sensor than the enzyme domain.

The term “domain” as used herein is a broad term, and is to be given itsordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), andrefers without limitation to regions of a membrane that can be layers,uniform or non-uniform gradients (i.e., anisotropic) or provided asportions of the membrane.

The term “electrical potential” as used herein is a broad term, and isto be given its ordinary and customary meaning to a person of ordinaryskill in the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to the electrical potentialdifference between two points in a circuit which is the cause of theflow of a current.

The terms “electrochemically reactive surface” and “electroactivesurface” as used herein are broad terms, and are to be given theirordinary and customary meaning to a person of ordinary skill in the art(and are not to be limited to a special or customized meaning), andrefer without limitation to the surface of an electrode where anelectrochemical reaction takes place. As one example, in a workingelectrode, H₂O₂ (hydrogen peroxide) produced by an enzyme-catalyzedreaction of an analyte being detected reacts and thereby creates ameasurable electric current. For example, in the detection of glucose,glucose oxidase produces H₂O₂ as a byproduct. The H₂O₂ reacts with thesurface of the working electrode to produce two protons (2H⁺), twoelectrons (2e⁻), and one molecule of oxygen (O₂), which produces theelectric current being detected. In the case of the counter electrode, areducible species, for example, O₂ is reduced at the electrode surfacein order to balance the current being generated by the workingelectrode.

The term “host” as used herein is a broad term, and is to be given itsordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), andrefers without limitation to animals (e.g., humans) and plants. In someexamples, a host can include domesticated animals (e.g., cats, dogs,etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), andbirds. In other examples, a host can include a mammal, such as a primateor a human.

The terms “interferents” and “interfering species” as used herein arebroad terms, and are to be given their ordinary and customary meaning toa person of ordinary skill in the art (and are not to be limited to aspecial or customized meaning), and refer without limitation to effectsor species that interfere with the measurement of an analyte of interestin a sensor to produce a signal that does not accurately represent theanalyte measurement. In an exemplary electrochemical sensor, interferingspecies can include compounds with an oxidation potential that overlapswith that of the analyte to be measured.

The terms “non-zwitterionic dipole” and “non-zwitterionic dipolarcompound” as used herein are broad terms, and are to be given theirordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), and referwithout limitation to compounds in which a neutral molecule of thecompound have a positive and negative electrical charge at differentlocations within the molecule. The positive and negative electricalcharges within the molecule can be any non-zero, but less than fullunit, charges.

The terms “operable connection,” “operably connected,” and “operablylinked” as used herein are broad terms, and are to be given theirordinary and customary meaning to a person of ordinary skill in the art(and are not to be limited to a special or customized meaning), andrefer without limitation to one or more components linked to anothercomponent(s) in a manner that allows transmission of signals between thecomponents. For example, one or more electrodes can be used to detectthe amount of analyte in a sample and convert that information into asignal; the signal can then be transmitted to a circuit. In this case,the electrode is “operably linked” to the electronic circuitry.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance can or cannot occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The term “polyampholytic polymer” as used herein is a broad term, and isto be given its ordinary and customary meaning to a person of ordinaryskill in the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to polymers comprising bothcationic and anionic groups. Such polymers can be prepared to have aboutequal numbers of positive and negative charges, and thus the surface ofsuch polymers can be about net neutrally charged. Alternatively, suchpolymers can be prepared to have an excess of either positive ornegative charges, and thus the surface of such polymers can be netpositively or negatively charged, respectively.

The term “polyzwitterions” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to polymers where a repeatingunit of the polymer chain is a zwitterionic moiety. Polyzwitterions arealso known as polybetaines. Since polyzwitterions have both cationic andanionic groups, they are a type of polyampholytic polymer. They areunique, however, because the cationic and anionic groups are both partof the same repeating unit, which means a polyzwitterion has the samenumber of cationic groups and anionic groups whereas otherpolyampholytic polymers can have more of one ionic group than the other.Also, polyzwitterions have the cationic group and anionic group as partof a repeating unit. Polyampholytic polymers need not have cationicgroups connected to anionic groups, they can be on different repeatingunits and thus may be distributed apart from one another at randomintervals, or one ionic group may outnumber the other.

The term “proximal to” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to the spatial relationshipbetween various elements in comparison to a particular point ofreference. For example, some embodiments of a device include a membranesystem having a biointerface layer and an enzyme layer. If the sensor isdeemed to be the point of reference and the enzyme layer is positionednearer to the sensor than the biointerface layer, then the enzyme layeris more proximal to the sensor than the biointerface layer.

The terms “raw data stream” and “data stream” as used herein are broadterms, and are to be given their ordinary and customary meaning to aperson of ordinary skill in the art (and are not to be limited to aspecial or customized meaning), and refer without limitation to ananalog or digital signal directly related to the measured glucoseconcentration from the glucose sensor. In one example, the raw datastream is digital data in “counts” converted by an A/D converter from ananalog signal (for example, voltage or amps) representative of a glucoseconcentration. The terms broadly encompass a plurality of time spaceddata points from a substantially continuous glucose sensor, whichcomprises individual measurements taken at time intervals ranging fromfractions of a second up to, for example, 1, 2, or 5 minutes or longer.

The terms “sensing membrane” and “membrane system” as used herein arebroad terms, and are to be given their ordinary and customary meaning toa person of ordinary skill in the art (and are not to be limited to aspecial or customized meaning), and refers without limitation to apermeable or semi-permeable membrane that can comprise one or moredomains or layers and constructed of materials of a few μm thickness ormore, which are permeable to oxygen and may or may not be permeable toan analyte of interest. In one example, the sensing membrane or membranesystem can comprise an immobilized glucose oxidase enzyme, which allowsan electrochemical reaction to occur to measure a concentration ofglucose.

The terms “sensing region,” “sensor,” and “sensing mechanism” as usedherein are broad terms, and are to be given their ordinary and customarymeaning to a person of ordinary skill in the art (and are not to belimited to a special or customized meaning), and refer withoutlimitation to the region or mechanism of a monitoring device responsiblefor the detection of a particular analyte.

The term “sensitivity” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to an amount signal (e.g., inthe form of electrical current and/or voltage produced by apredetermined amount (unit) of the measured analyte. For example, in oneembodiment, a sensor has a sensitivity (or slope) of from about 1 toabout 100 picoAmps of current for every 1 mg/dL of glucose analyte.

The terms “zwitterion” and “zwitterionic compound” as used herein arebroad terms, and are to be given their ordinary and customary meaning toa person of ordinary skill in the art (and is not to be limited to aspecial or customized meaning), and refer without limitation tocompounds in which a neutral molecule of the compound has a unitpositive and unit negative electrical charge at different locationswithin the molecule. Such compounds are a type of dipolar compounds, andare also sometimes referred to as “inner salts.”

A “zwitterion precursor” or “zwitterionic compound precursor” as usedherein are broad terms, and are to be given their ordinary and customarymeaning to a person of ordinary skill in the art (and is not to belimited to a special or customized meaning), and refer withoutlimitation to any compound that is not itself a zwitterion, but canbecome a zwitterion in a final or transition state through chemicalreaction. In some embodiments described herein, devices comprisezwitterion precursors that can be converted to zwitterions prior to invivo implantation of the device. Alternatively, in some embodimentsdescribed herein, devices comprise zwitterion precursors that can beconverted to zwitterions by some chemical reaction that occurs after invivo implantation of the device. Such reactions are known to the skilledin art and include ring opening reaction, addition reaction such asMichael addition. This method is especially useful when thepolymerization of betaine containing monomer is difficult due totechnical challenges such as solubility of betaine monomer to achievedesired physical properties such as molecular weight and mechanicalstrength. Post-polymerization modification or conversion of betaineprecursor can be a practical way to achieve desired polymer structureand composition. Examples of such as precursors include tertiary amines,quaternary amines, pyridines, and others detailed herein.

A “zwitterion derivative” or “zwitterionic compound derivative” as usedherein are broad terms, and are to be given their ordinary and customarymeaning to a person of ordinary skill in the art (and is not to belimited to a special or customized meaning), and refer withoutlimitation to any compound that is not itself a zwitterion, but ratheris the product of a chemical reaction where a zwitterion is converted toa non-zwitterion. Such reactions can be reversible, such that undercertain conditions zwitterion derivatives can act as zwitterionprecursors. For example, hydrolyzable betaine esters formed fromzwitterionic betaines are cationic zwitterion derivatives that under theappropriate conditions are capable of undergoing hydrolysis to revert tozwitterionic betaines.

As employed herein, the following abbreviations apply: Eq and Eqs(equivalents); mEq (milliequivalents); M (molar); mM (millimolar) μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); Kg (kilograms); L (liters); mL (milliliters); dL(deciliters); μL (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); h and hr (hours); min (minutes); s andsec (seconds); ° C. (degrees Centigrade).

Sensor System

FIG. 1 is a schematic of a continuous analyte sensor system 100 attachedto a host and communicating with a number of other example devices110-113. A transcutaneous analyte sensor system comprising an on-skinsensor assembly 600 is shown which is fastened to the skin of a host viaa disposable housing (not shown). The system includes a transcutaneousanalyte sensor 200 and an electronics unit (referred to interchangeablyas “sensor electronics” or “transmitter”) 500 for wirelesslytransmitting analyte information to a receiver. During use, a sensingportion of the sensor 200 is under the host's skin and a contact portionof the sensor 200 is operably connected (e.g., electrically connected)to the electronics unit 500. The electronics unit 500 is engaged with ahousing which is attached to an adhesive patch fastened to the skin ofthe host.

The on-skin sensor assembly 600 may be attached to the host with use ofan applicator adapted to provide convenient and secure application. Suchan applicator may also be used for inserting the sensor 200 through thehost's skin. Once the sensor 200 has been inserted, the applicatordetaches from the sensor assembly.

In general, the continuous analyte sensor system 100 includes any sensorconfiguration that provides an output signal indicative of aconcentration of an analyte. The output signal including (e.g., sensordata, such as a raw data stream, filtered data, smoothed data, and/orotherwise transformed sensor data) is sent to receiver which may bee.g., a smart phone, smart watch, dedicated device and the like. In oneembodiment, the analyte sensor system 100 includes a transcutaneousglucose sensor, such as is described in US Patent Publication No.US-2011-0027127-A1, the contents of which is hereby incorporated byreference in its entirety. In some embodiments, the sensor system 100includes a continuous glucose sensor and comprises a transcutaneoussensor such as described in U.S. Pat. No. 6,565,509 to Say et al., forexample. In another embodiment, the sensor system 100 includes acontinuous glucose sensor and comprises a subcutaneous sensor such asdescribed with reference to U.S. Pat. No. 6,579,690 to Bonnecaze et al.or U.S. Pat. No. 6,484,046 to Say et al., for example. In anotherembodiment, the sensor system 100 includes a continuous glucose sensorand comprises a subcutaneous sensor such as described with reference toU.S. Pat. No. 6,512,939 to Colvin et al. In another embodiment, thesensor system 100 includes a continuous glucose sensor and comprises anintravascular sensor such as described with reference to U.S. Pat. No.6,477,395 to Schulman et al., for example. In another embodiment, thesensor system 100 includes a continuous glucose sensor and comprises anintravascular sensor such as described with reference to U.S. Pat. No.6,424,847 to Mastrototaro et al. Other signal processing techniques andglucose monitoring system embodiments suitable for use with theembodiments described herein are described in U.S. Patent PublicationNo. US-2005-0203360-A1 and U.S. Patent Publication No.US-2009-0192745-A1, the contents of which are hereby incorporated byreference in their entireties. The sensor extends through a housing,which maintains the sensor on the skin and provides for electricalconnection of the sensor to sensor electronics, provided in theelectronics unit.

In one embodiment, the sensor is formed from a wire or is in a form of awire. For example, the sensor can include an elongated conductive body,such as a bare elongated conductive core (e.g., a metal wire) or anelongated conductive core coated with one, two, three, four, five, ormore layers of material, each of which may or may not be conductive. Theelongated sensor may be long and thin, yet flexible and strong. Forexample, in some embodiments, the smallest dimension of the elongatedconductive body is less than about 0.1 inches (0.3 cm), less than about0.075 inches (0.20 cm), less than about 0.05 inches (0.13 cm), less thanabout 0.025 inches (0.06 cm), less than about 0.01 inches (0.03 cm),less than about 0.004 inches (0.01 cm), or less than about 0.002 inches(0.005 cm). The sensor may have a circular cross-section. In someembodiments, the cross-section of the elongated conductive body can beovoid, rectangular, triangular, polyhedral, star-shaped, C-shaped,T-shaped, X-shaped, Y-Shaped, irregular, or the like. In one embodiment,a conductive wire electrode is employed as a core. To such a cladelectrode, one or two additional conducting layers may be added (e.g.,with intervening insulating layers provided for electrical isolation).The conductive layers can be comprised of any suitable material. Incertain embodiments, it can be desirable to employ a conductive layercomprising conductive particles (i.e., particles of a conductivematerial) in a polymer or other binder.

In certain embodiments, the materials used to form the elongatedconductive body (e.g., stainless steel, titanium, tantalum, platinum,platinum-iridium, iridium, certain polymers, and/or the like) can bestrong and hard, and therefore are resistant to breakage. For example,in some embodiments, the ultimate tensile strength of the elongatedconductive body is from about 80 kPsi to about 500 kPsi. In anotherexample, in some embodiments, the Young's modulus of the elongatedconductive body is from about 160 GPa to about 220 GPa. In still anotherexample, in some embodiments, the yield strength of the elongatedconductive body is from about 60 kPsi to about 2200 kPsi. In someembodiments, the sensor's small diameter provides (e.g., imparts,enables) flexibility to these materials, and therefore to the sensor asa whole. Thus, the sensor can withstand repeated forces applied to it bysurrounding tissue.

In addition to providing structural support, resiliency and flexibility,in some embodiments, the core (or a component thereof) provideselectrical conduction for an electrical signal from the workingelectrode to sensor electronics (not shown). In some embodiments, thecore comprises a conductive material, such as stainless steel, titanium,tantalum, a conductive polymer, and/or the like. However, in otherembodiments, the core is formed from a non-conductive material, such asa non-conductive polymer. In yet other embodiments, the core comprises aplurality of layers of materials. For example, in one embodiment thecore includes an inner core and an outer core. In a further embodiment,the inner core is formed of a first conductive material and the outercore is formed of a second conductive material. For example, in someembodiments, the first conductive material is stainless steel, titanium,tantalum, a conductive polymer, an alloy, and/or the like, and thesecond conductive material is conductive material selected to provideelectrical conduction between the core and the first layer, and/or toattach the first layer to the core (e.g., if the first layer is formedof a material that does not attach well to the core material). Inanother embodiment, the core is formed of a non-conductive material(e.g., a non-conductive metal and/or a non-conductive polymer) and thefirst layer is a conductive material, such as stainless steel, titanium,tantalum, a conductive polymer, and/or the like. The core and the firstlayer can be of a single (or same) material, e.g., platinum. One skilledin the art appreciates that additional configurations are possible.

In the illustrated embodiments, the electronics unit 500 is releasablyattachable to the sensor 200. The electronics unit 500 includeselectronic circuitry associated with measuring and processing thecontinuous analyte sensor data, and is configured to perform algorithmsassociated with processing and calibration of the sensor data. Forexample, the electronics unit 500 can provide various aspects of thefunctionality of a sensor electronics module as described in U.S. PatentPublication No. US-2009-0240120-A1 and U.S. patent application Ser. No.13/247,856 filed Sep. 28, 2011 and entitled “ADVANCED CONTINUOUS ANALYTEMONITORING SYSTEM,” the contents of which are hereby incorporated byreference in their entireties. The electronics unit 500 may includehardware, firmware, and/or software that enable measurement of levels ofthe analyte via a glucose sensor, such as an analyte sensor 200. Forexample, the electronics unit 500 can include a potentiostat, a powersource for providing power to the sensor 200, other components usefulfor signal processing and data storage, and preferably a telemetrymodule for one- or two-way data communication between the electronicsunit 500 and one or more receivers, repeaters, and/or display devices,such as devices 110-113. Electronics can be affixed to a printed circuitboard (PCB), or the like, and can take a variety of forms. For example,the electronics can take the form of an integrated circuit (IC), such asan Application-Specific Integrated Circuit (ASIC), a microcontroller,and/or a processor. The electronics unit 500 may include sensorelectronics that are configured to process sensor information, such asstoring data, analyzing data streams, calibrating analyte sensor data,estimating analyte values, comparing estimated analyte values with timecorresponding measured analyte values, analyzing a variation ofestimated analyte values, and the like. Examples of systems and methodsfor processing sensor analyte data are described in more detail hereinand in U.S. Pat. Nos. 7,310,544, 6,931,327, U.S. Patent Publication No.2005-0043598-A1, U.S. Patent Publication No. 2007-0032706-A1, U.S.Patent Publication No. 2007-0016381-A1, U.S. Patent Publication No.2008-0033254-A1, U.S. Patent Publication No. 2005-0203360-A1, U.S.Patent Publication No. 2005-0154271-A1, U.S. Patent Publication No.2005-0192557-A1, U.S. Patent Publication No. 2006-0222566-A1, U.S.Patent Publication No. 2007-0203966-A1 and U.S. Patent Publication No.2007-0208245-A1, the contents of which are hereby incorporated byreference in their entireties.

One or more repeaters, receivers and/or display devices, such as key fobrepeater 110, medical device receiver 111 (e.g., insulin delivery deviceand/or dedicated glucose sensor receiver), smart phone 112, portablecomputer 113, and the like are operatively linked to the electronicsunit, which receive data from the electronics unit 500, which is alsoreferred to as the transmitter and/or sensor electronics body herein,and in some embodiments transmit data to the electronics unit 500. Forexample, the sensor data can be transmitted from the sensor electronicsunit 500 to one or more of key fob repeater 110, medical device receiver111, smart phone 112, portable computer 113, and the like. In oneembodiment, a display device includes an input module with a quartzcrystal operably connected to an RF transceiver (not shown) thattogether function to transmit, receive and synchronize data streams fromthe electronics unit 500. However, the input module can be configured inany manner that is capable of receiving data from the electronics unit500. Once received, the input module sends the data stream to aprocessor that processes the data stream, such as described in moredetail below. The processor is the central control unit that performsthe processing, such as storing data, analyzing data streams,calibrating analyte sensor data, estimating analyte values, comparingestimated analyte values with time corresponding measured analytevalues, analyzing a variation of estimated analyte values, downloadingdata, and controlling the user interface by providing analyte values,prompts, messages, warnings, alarms, and the like. The processorincludes hardware that performs the processing described herein, forexample read-only memory (ROM) provides permanent or semi-permanentstorage of data, storing data such as sensor ID (sensor identity),receiver ID (receiver identity), and programming to process data streams(for example, programming for performing estimation and other algorithmsdescribed elsewhere herein) and random access memory (RAM) stores thesystem's cache memory and is helpful in data processing. An outputmodule, which may be integral with and/or operatively connected with theprocessor, includes programming for generating output based on thesensor data received from the electronics unit (and any processing thatincurred in the processor).

In some embodiments, analyte values are displayed on a display device.In some embodiments, prompts or messages can be displayed on the displaydevice to convey information to the user, such as reference outliervalues, requests for reference analyte values, therapy recommendations,deviation of the measured analyte values from the estimated analytevalues, or the like. Additionally, prompts can be displayed to guide theuser through calibration or trouble-shooting of the calibration.

Additionally, data output from the output module can provide wired orwireless, one- or two-way communication between the receiver and anexternal device. The external device can be any device that interfacesor communicates with the receiver. In some embodiments, the externaldevice is a computer, and the receiver is able to download current orhistorical data for retrospective analysis by a physician, for example.In some embodiments, the external device is a modem, and the receiver isable to send alerts, warnings, emergency messages, or the like, viatelecommunication lines to another party, such as a doctor or familymember. In some embodiments, the external device is an insulin pen, andthe receiver is able to communicate therapy recommendations, such asinsulin amount and time, to the insulin pen. In some embodiments, theexternal device is an insulin pump, and the receiver is able tocommunicate therapy recommendations, such as insulin amount and time tothe insulin pump. The external device can include other technology ormedical devices, for example pacemakers, implanted analyte sensorpatches, other infusion devices, telemetry devices, or the like. Thereceiver may communicate with the external device, and/or any number ofadditional devices, via any suitable communication protocol, includingradio frequency, Bluetooth, universal serial bus, any of the wirelesslocal area network (WLAN) communication standards, including the IEEE802.11, 802.15, 802.20, 802.22 and other 802 communication protocols,ZigBee, wireless (e.g., cellular) telecommunication, paging networkcommunication, magnetic induction, satellite data communication, GPRS,ANT, and/or a proprietary communication protocol.

The implementations described herein generally discuss sensorsconstituted by one or more sensor wires. However, it will be understoodthat the sensors are not limited to such wire shaped or lineararrangements. Rather, the sensors may be implemented as planar sensors,volumetric sensors, point sensors, or in other shapes as will beunderstood given this description.

Membrane Systems

Membrane systems disclosed herein are suitable for use with implantabledevices in contact with a biological fluid. For example, the membranesystems can be utilized with implantable devices, such as devices formonitoring and determining analyte levels in a biological fluid, forexample, devices for monitoring glucose levels for individuals havingdiabetes. In some embodiments, the analyte-measuring device is acontinuous device. The analyte-measuring device can employ any suitablesensing element to provide the raw signal, including but not limited tothose involving enzymatic, chemical, physical, electrochemical,spectrophotometric, polarimetric, calorimetric, radiometric,immunochemical, or like elements.

Although some of the description that follows is directed atglucose-measuring devices, including the described membrane systems andmethods for their use, these membrane systems are not limited to use indevices that measure or monitor glucose. These membrane systems aresuitable for use in any of a variety of devices, including, for example,devices that detect and quantify other analytes present in biologicalfluids (e.g. cholesterol, amino acids, alcohol, galactose, and lactate),cell transplantation devices (see, for example, U.S. Pat. Nos.6,015,572, 5,964,745, and 6,083,523), drug delivery devices (see, forexample, U.S. Pat. Nos. 5,458,631, 5,820,589, and 5,972,369), and thelike, which are incorporated herein by reference in their entireties fortheir teachings of membrane systems.

In one embodiment, the analyte-measuring device is an implantableglucose sensor, such as described with reference to U.S. Pat. No.6,001,067 and U.S. Patent Publication No. US-2005-0027463-A1, which areincorporated herein by reference in their entireties. In anotherembodiment, the analyte-measuring device is a glucose sensor, such asdescribed with reference to U.S. Patent Publication No.US-2006-0020187-A1, which is incorporated herein by reference in itsentirety. In still other embodiments, the sensor is configured to beimplanted in a host vessel or extra-corporeally, such as is described inU.S. Patent Publication No. US-2007-0027385-A1, U.S. Patent PublicationNo. US-2008-0119703-A1, U.S. Patent Publication No. US-2008-0108942-A1,and U.S. Patent Publication No. US-2007-0197890-A1, which areincorporated herein by reference in their entirety. In some embodiments,the sensor is configured as a dual-electrode sensor, such as describedin U.S. Patent Publication No. US-2005-0143635-A1, U.S. PatentPublication No. US-2007-0027385-A1, U.S. Patent Publication No.US-2007-0213611-A1, and U.S. Patent Publication No. US-2008-0083617-A1,which are incorporated herein by reference in their entirety. In onealternative embodiment, the continuous glucose sensor comprises a sensorsuch as described in U.S. Pat. No. 6,565,509 to Say et al., for example.In another alternative embodiment, the continuous glucose sensorcomprises a subcutaneous sensor such as described with reference to U.S.Pat. No. 6,579,690 to Bonnecaze et al. or U.S. Pat. No. 6,484,046 to Sayet al., for example. In another alternative embodiment, the continuousglucose sensor comprises a refillable subcutaneous sensor such asdescribed with reference to U.S. Pat. No. 6,512,939 to Colvin et al.,for example. In yet another alternative embodiment, the continuousglucose sensor comprises an intravascular sensor such as described withreference to U.S. Pat. No. 6,477,395 to Schulman et al., for example. Inanother alternative embodiment, the continuous glucose sensor comprisesan intravascular sensor such as described with reference to U.S. Pat.No. 6,424,847 to Mastrototaro et al. In some embodiments, the electrodesystem can be used with any of a variety of known in vivo analytesensors or monitors, such as U.S. Pat. No. 7,157,528 to Ward; U.S. Pat.No. 6,212,416 to Ward et al.; U.S. Pat. No. 6,119,028 to Schulman etal.; U.S. Pat. No. 6,400,974 to Lesho; U.S. Pat. No. 6,595,919 to Berneret al.; U.S. Pat. No. 6,141,573 to Kurnik et al.; U.S. Pat. No.6,122,536 to Sun et al.; European Patent Publication No. EP 1153571 toVarall et al.; U.S. Pat. No. 6,512,939 to Colvin et al.; U.S. Pat. No.5,605,152 to Slate et al.; U.S. Pat. No. 4,431,004 to Bessman et al.;U.S. Pat. No. 4,703,756 to Gough et al.; U.S. Pat. No. 6,514,718 toHeller et al.; U.S. Pat. No. 5,985,129 to Gough et al.; PCTInternational Publication No. WO4/021877 to Caduff; U.S. Pat. No.5,494,562 to Maley et al.; U.S. Pat. No. 6,120,676 to Heller et al.; andU.S. Pat. No. 6,542,765 to Guy et al., which are incorporated byreference herein in their entirities. In general, it is understood thatthe disclosed embodiments are applicable to a variety of continuousanalyte measuring device configurations.

In some embodiments, a long term sensor (e.g., wholly implantable orintravascular) is configured and arranged to function for a time periodof from about 30 days or less to about one year or more (e.g., a sensorsession). In some embodiments, a short term sensor (e.g., one that istranscutaneous or intravascular) is configured and arranged to functionfor a time period of from about a few hours to about 30 days, includinga time period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 days (e.g.,a sensor session). As used herein, the term “sensor session” is a broadterm and refers without limitation to the period of time the sensor isapplied to (e.g., implanted in) the host or is being used to obtainsensor values. For example, in some embodiments, a sensor sessionextends from the time of sensor implantation (e.g., including insertionof the sensor into subcutaneous tissue and placing the sensor into fluidcommunication with a host's circulatory system) to the time when thesensor is removed.

In general, the membrane system includes a plurality of domains, forexample, an electrode domain, an interference domain, an enzyme domain,a resistance domain, and a biointerface domain. The membrane system canbe deposited on the exposed electroactive surfaces using known thin filmtechniques (for example, vapor deposition, spraying, electrodepositing,dipping, brush coating, film coating, drop-let coating, and the like).Addition steps may be applied following the membrane materialdeposition, for example, drying, annealing, and curing (for example, UVcuring, thermal curing, moisture curing, radiation curing, and the like)to enhance certain properties such as mechanical properties, signalstability, and selectivity. In a typical process, upon deposition ofresistance layer membrane, a biointerface layer having a “dry film”thickness of from about 0.05 μm, or less, to about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16 μm. “Dry film” thickness refers tothe thickness of a cured film cast from a coating formulation bystandard coating techniques.

In certain embodiments, the biointerface layer is formed of abiointerface polymer, wherein the biointerface polymer comprisespolyurethane and/or polyurea segments and one or more zwitterionicrepeating units. In some embodiments, the biointerface layer coatingsare formed of a polyurethane urea having carboxyl betaine groupsincorporated in the polymer and non-ionic hydrophilic polyethylene oxidesegments, wherein the polyurethane urea polymer is dissolved in organicor non-organic solvent system according to a pre-determined coatingformulation, and is crosslinked with an isocyanate crosslinker and curedat moderate temperature of about 50° C. The solvent system can be asingle solvent or a mixture of solvents to aid the dissolution ordispersion of the polymer. The solvents can be the ones selected as thepolymerization media or added after polymerization is completed. Thesolvents are preferably selected from the ones having lower boilingpoint to facilitate drying and lower in toxicity for implantapplications. Examples of these solvent includes aliphatic ketone,ester, ether, alcohol, hydrocarbons, and the likes. Depending on thefinal thickness of the biointerface layer and solution viscosity (asrelated to the percent of polymer solid), the coating can be applied ina single step or multiple repeated steps of the chosen process such asdipping to build the desired thickness. Yet in other embodiments, thebiointerface polymers are formed of a polyurethane urea havingcarboxylic acid groups and carboxyl betaine groups incorporated in thepolymer and non-ionic hydrophilic polyethylene oxide segments, whereinthe polyurethane urea polymer is dissolved in organic or non-organicsolvent system in a coating formulation, and is crosslinked with an acarbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)and cured at moderate temperature of about 50° C.

In certain embodiments, the biointerface layer is formed of abiointerface polymer. The biointerface polymer is a polyzwitterion. Thebiointerface polymer may also comprise polyurethane and/or polyureasegments. For example, the biointerface polymer can comprise apolyurethane copolymer such as polyether-urethane-urea,polycarbonate-urethane, polyether-urethane, silicone-polyether-urethane,silicone-polycarbonate-urethane, polyester-urethane, polyurethane-urea,and the like. Since these polyurethane and/or polyurea segments containurea and/or urethane bonds formed from polyisocyanate and short chainpolyol or polyamine, which are hydrogen bonding rich moieties, thesesegments are referred to herein as “hard segments.” These segments canalso be relatively hydrophobic. In addition to polyurethane and/orpolyurea hard segments, the disclosed biointerface polymers can alsocomprise soft segments, which have relatively poor hydrogen bonding.Soft segment are usually composed of polyols of polycarbonates,polyesters, polyethers, polyarylene, and polyalkylene, and the like. Thesoft segments can be either hydrophobic or hydrophilic. In certainembodiments, the biointerface layer is formed of a biointerface polymer,wherein the biointerface polymer comprises polyurethane and/or polyureasegments and one or more zwitterionic repeating units. In someembodiments, the biointerface polymer is formed of a polyurethane ureahaving betaine groups incorporated in the polymer and non-ionichydrophilic polyethylene oxide segments.

In other embodiments, the biointerface layer coatings are formed of apolyurethane urea having sulfo betaine groups incorporated in thepolymer and non-ionic hydrophilic polyethylene oxide segments, whereinthe polyurethane urea polymer is dissolved in organic or non-organicsolvent system according to a pre-determined coating formulation, and iscrosslinked with an isocyanate crosslinker and cured at moderatetemperature of about 50° C. The solvent system can be a single solventor a mixture of solvents to aid the dissolution or dispersion of thepolymer. The solvents can be the ones selected as the polymerizationmedia or added after polymerization is completed. The solvents arepreferably selected from the ones having lower boiling point tofacilitate drying and lower in toxicity for implant applications.Examples of these solvent includes aliphatic ketone, ester, ether,alcohol, hydrocarbons, and the likes. Depending on the final thicknessof the biointerface layer and solution viscosity (as related to thepercent of polymer solid), the coating can be applied in a single stepor multiple repeated steps of the chosen process such as dipping tobuild the desired thickness. Yet in other embodiments, the biointerfacepolymers are formed of a polyurethane urea having unsaturatedhydrocarbon groups and sulfo betaine groups incorporated in the polymerand non-ionic hydrophilic polyethylene oxide segments, wherein thepolyurethane urea polymer is dissolved in organic or non-organic solventsystem in a coating formulation, and is crosslinked in the presence ofinitiators with heat or irradiation including UV, LED light, electronbeam, and the likes, and cured at moderate temperature of about 50° C.Examples of unsaturated hydrocarbon includes allyl groups, vinyl groups,acrylate, methacrylate, alkenes, alkynes, and the likes.

In certain embodiments, the enzyme layer is formed of an enzyme layerpolymer and an active enzyme, wherein the enzyme layer polymer comprisespolyurethane and/or polyurea segments and one or more zwitterionicrepeating units. In some embodiments, the enzyme layer coatings areformed of a polyurethane urea having carboxyl betaine groupsincorporated in the polymer and non-ionic hydrophilic polyethylene oxidesegments, wherein the polyurethane urea polymer is dissolved in organicor non-organic solvent system according to a pre-determined coatingformulation, and is crosslinked with an isocyanate crosslinker and curedat moderate temperature of about 50° C. The solvent system can be asingle solvent or a mixture of solvents to aid the dissolution ordispersion of the polymer. The solvents can be the ones selected as thepolymerization media or added after polymerization is completed. Thesolvents are preferably selected from the ones having lower boilingpoint to facilitate drying, having a lower potential to denature theenzyme, and lower in toxicity for implant applications. Examples ofthese solvents include water, aliphatic ketone, ester, ether, alcohol,hydrocarbons, and the likes. Depending on the final thickness of theenzyme layer and solution viscosity (as related to the percent ofpolymer solid), the coating can be applied in a single step or multiplerepeated steps of the chosen process such as dipping to build thedesired thickness. Yet in other embodiments, the enzyme layer polymersare formed of a polyurethane urea having carboxylic acid groups andcarboxyl betaine groups incorporated in the polymer and non-ionichydrophilic polyethylene oxide segments, wherein the polyurethane ureapolymer is dissolved in organic or non-organic solvent system in acoating formulation, and is crosslinked with an a carbodiimide (e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and cured atmoderate temperature of about 50° C. Other crosslinkers can be used aswell, such as polyfunctional aziridines.

In other embodiments, the enzyme layer is formed of a polyurethane ureahaving sulfo betaine groups incorporated in the polymer and non-ionichydrophilic polyethylene oxide segments, wherein the polyurethane ureapolymer is dissolved in organic or non-organic solvent system accordingto a pre-determined coating formulation, and is crosslinked with anisocyanate crosslinker and cured at moderate temperature of about 50° C.The solvent system can be a single solvent or a mixture of solvents toaid the dissolution or dispersion of the polymer. The solvents can bethe ones selected as the polymerization media or added afterpolymerization is completed. The solvents are preferably selected fromthe ones having lower boiling point to facilitate drying and lower intoxicity for implant applications. Examples of these solvent includesaliphatic ketone, ester, ether, alcohol, hydrocarbons, and the likes.Depending on the final thickness of the enzyme layer and solutionviscosity (as related to the percent of polymer solid), the coating canbe applied in a single step or multiple repeated steps of the chosenprocess such as dipping to build the desired thickness. Yet in otherembodiments, the enzyme layer polymers are formed of a polyurethane ureahaving unsaturated hydrocarbon groups and sulfo betaine groupsincorporated in the polymer and non-ionic hydrophilic polyethylene oxidesegments, wherein the polyurethane urea polymer is dissolved in organicor non-organic solvent system in a coating formulation, and iscrosslinked in the presence of initiators with heat or irradiationincluding UV, LED light, electron beam, and the like, and cured atmoderate temperature of about 50° C. Examples of unsaturated hydrocarbonincludes allyl groups, vinyl groups, acrylate, methacrylate, alkenes,alkynes, and the likes.

FIGS. 3A through 3C illustrate an embodiment of the in vivo portion of acontinuous analyte sensor 400, which includes an elongated conductivebody 402. The elongated conductive body 402 includes a core 410 (seeFIG. 3B) and a first layer 412 at least partially surrounding the core.The first layer includes a working electrode (for example, located inwindow 406) and a membrane 408 located over the working electrode. Insome embodiments, the core and first layer can be of a single material(such as, for example, platinum). In some embodiments, the elongatedconductive body is a composite of at least two materials, such as acomposite of two conductive materials, or a composite of at least oneconductive material and at least one non-conductive material. In someembodiments, the elongated conductive body comprises a plurality oflayers. In certain embodiments, there are at least two concentric orannular layers, such as a core formed of a first material and a firstlayer formed of a second material. However, additional layers can beincluded in some embodiments. In some embodiments, the layers arecoaxial.

The elongated conductive body can be long and thin, yet flexible andstrong. For example, in some embodiments, the smallest dimension of theelongated conductive body is less than about 0.1 inches, 0.075 inches,0.05 inches, 0.025 inches, 0.01 inches, 0.004 inches, or 0.002 inches.While the elongated conductive body is illustrated in FIGS. 3A through3C as having a circular cross-section, in other embodiments thecross-section of the elongated conductive body can be ovoid,rectangular, triangular, polyhedral, star-shaped, C-shaped, T-shaped,X-shaped, Y-Shaped, irregular, or the like. In one embodiment, aconductive wire electrode is employed as a core. To such a cladelectrode, two additional conducting layers can be added (e.g., withintervening insulating layers provided for electrical isolation). Theconductive layers can be comprised of any suitable material. In certainembodiments, it can be desirable to employ a conductive layer comprisingconductive particles (i.e., particles of a conductive material) in apolymer or other binder.

The materials used to form the elongated conductive body (such as, forexample, stainless steel, titanium, tantalum, platinum,platinum-iridium, iridium, certain polymers, and/or the like) can bestrong and hard, and therefore are resistant to breakage. In someembodiments, the sensor's small diameter provides flexibility to thesematerials, and therefore to the sensor as a whole. Thus, the sensor canwithstand repeated forces applied to it by surrounding tissue.

In addition to providing structural support, resiliency and flexibility,in some embodiments, the core 410, or a component thereof, provideselectrical conduction for an electrical signal from the workingelectrode to sensor electronics (not shown). In some embodiments, thecore 410 comprises a conductive material, such as stainless steel,titanium, tantalum, a conductive polymer, and/or the like. However, inother embodiments, the core is formed from a non-conductive material,such as a non-conductive polymer. In yet other embodiments, the corecomprises a plurality of layers of materials. For example, in oneembodiment the core includes an inner core and an outer core. In afurther embodiment, the inner core is formed of a first conductivematerial and the outer core is formed of a second conductive material.For example, in some embodiments, the first conductive material isstainless steel, titanium, tantalum, a conductive polymer, an alloy,and/or the like, and the second conductive material is a conductivematerial selected to provide electrical conduction between the core andthe first layer, and/or to attach the first layer to the core (that is,if the first layer is formed of a material that does not attach well tothe core material). In another embodiment, the core is formed of anon-conductive material (such as, for example, a non-conductive metaland/or a non-conductive polymer) and the first layer is formed of aconductive material, such as stainless steel, titanium, tantalum, aconductive polymer, and/or the like. The core and the first layer can beof a single (or same) material, such as platinum. One skilled in the artappreciates that additional configurations are possible.

Referring again to FIGS. 3A through 3C, the first layer 412 can beformed of a conductive material and the working electrode can be anexposed portion of the surface of the first layer 412. Accordingly, thefirst layer 412 can be formed of a material configured to provide asuitable electroactive surface for the working electrode, a materialsuch as, but not limited to, platinum, platinum-iridium, gold,palladium, iridium, graphite, carbon, a conductive polymer, an alloyand/or the like.

As illustrated in FIG. 3B and FIG. 3C, a second layer 404 surrounds atleast a portion of the first layer 412, thereby defining the boundariesof the working electrode. In some embodiments, the second layer 404serves as an insulator and is formed of an insulating material, such aspolyimide, polyurethane, parylene, or any other known insulatingmaterials. For example, in one embodiment the second layer is disposedon the first layer and configured such that the working electrode isexposed via window 406. In some embodiments, an elongated conductivebody, including the core, the first layer and the second layer, isprovided. A portion of the second layer can be removed to form a window406, through which the electroactive surface of the working electrode(that is, the exposed surface of the first layer 412) is exposed. Insome embodiments, a portion of the second and (optionally) third layerscan be removed to form the window 406, thus exposing the workingelectrode. Removal of coating materials from one or more layers of theelongated conductive body (for example, to expose the electroactivesurface of the working electrode) can be performed by hand, excimerlasing, chemical etching, laser ablation, grit-blasting, or the like.

The sensor can further comprise a third layer 414 comprising aconductive material. For example, the third layer 414 can comprise areference electrode, which can be formed of a silver-containing materialthat is applied onto the second layer 404 (that is, the insulator).

The elongated conductive body 402 can further comprise one or moreintermediate layers (not shown) located between the core 410 and thefirst layer 412. For example, the intermediate layer can be one or moreof an insulator, a conductor, a polymer, and/or an adhesive.

It is contemplated that the ratio between the thickness of thesilver/silver chloride layer and the thickness of an insulator (such as,for example, polyurethane or polyimide) layer can be controlled, so asto allow for a certain error margin (that is, an error margin associatedwith the etching process) that would not result in a defective sensor(for example, due to a defect resulting from an etching process thatcuts into a depth more than intended, thereby unintentionally exposingan electroactive surface). This ratio can be different depending on thetype of etching process used, whether it is laser ablation, gritblasting, chemical etching, or some other etching method. In oneembodiment in which laser ablation is performed to remove asilver/silver chloride layer and a polyurethane layer, the ratio of thethickness of the silver/silver chloride layer and the thickness of thepolyurethane layer can be from about 1:5 to about 1:1, or from about 1:3to about 1:2.

In some embodiments, the core 410 comprises a non-conductive polymer andthe first layer 412 comprises a conductive material. Such a sensorconfiguration can advantageously provide reduced material costs, in thatit replaces a typically expensive material with an inexpensive material.For example, the core 410 can be formed of a non-conductive polymer,such as, a nylon or polyester filament, string or cord, which can becoated and/or plated with a conductive material, such as platinum,platinum-iridium, gold, palladium, iridium, graphite, carbon, aconductive polymer, and allows or combinations thereof.

As illustrated in FIG. 3C and FIG. 3D, the sensor can also include amembrane 408, such as those discussed elsewhere herein, for example,with reference to FIGS. 2A through 2C. The membrane 408 can include anenzyme layer (not shown), as described elsewhere herein. For example,the enzyme layer can include a catalyst or enzyme configured to reactwith an analyte. For example, the enzyme layer can be an immobilizedenzyme layer including glucose oxidase. In other embodiments, the enzymelayer can be impregnated with other oxidases, including, for example,galactose oxidase, cholesterol oxidase, amino acid oxidase, alcoholoxidase, lactate oxidase, or uricase.

FIG. 3B is a schematic illustrating an embodiment of an elongatedconductive body 402, or elongated body, wherein the elongated conductivebody is formed from at least two materials and/or layers of conductivematerial, as described in greater detail elsewhere herein. The term“electrode” can be used herein to refer to the elongated conductivebody, which includes the electroactive surface that detects the analyte.In some embodiments, the elongated conductive body provides anelectrical connection between the electroactive surface (that is, theworking electrode) and the sensor electronics (not shown). In certainembodiments, each electrode (that is, the elongated conductive body onwhich the electroactive surface is located) is formed from a fine wirewith a diameter of from about 0.001 inches (0.003 cm) or less to about0.01 inches (0.03 cm) or more. Each electrode can be formed from, forexample, a plated insulator, a plated wire, or bulk electricallyconductive material. For example, in some embodiments, the wire and/orelongated conductive body used to form a working electrode is about0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.015,0.02, 0.025, 0.03, 0.035, 0.04 or 0.045 inches in diameter.

Furthermore, the first layer can comprise an electroactive surface (thatis, the portion exposed through the window 406). The exposedelectroactive surface can be the working electrode. For example, if thesensor is an enzymatic electrochemical analyte sensor, the analyteenzymatically reacts with an enzyme in the membrane covering at least aportion of the electroactive surface. The reaction can generateelectrons (e⁻) that are detected at the electroactive surface as ameasurable electronic current. For example, in the detection of glucosewherein glucose oxidase produces hydrogen peroxide as a byproduct,hydrogen peroxide reacts with the surface of the working electrodeproducing two protons (2H⁺), two electrons (2e⁻) and one molecule ofoxygen (O₂), which produces the electronic current being detected.

As previously described with reference to FIG. 3A and as illustrated inFIG. 3C, an insulator 404 is disposed on at least a portion of theelongated conductive body 402. In some embodiments, the sensor isconfigured and arranged such that the elongated body includes a core 410and a first layer 412, and a portion of the first layer 412 is exposedvia window 406 in the insulator 404. In other embodiments, the sensor isconfigured and arranged such that the elongated body 402 includes a core410 embedded in an insulator 404, and a portion of the core 410 isexposed via the window 406 in the insulator 404. For example, theinsulating material can be applied to the elongated body 402 (by, forexample, screen-, ink-jet and/or block-print) in a configurationdesigned to leave at least a portion of the first layer's 412 surface(or the core's 410 surface) exposed. For example, the insulatingmaterial can be printed in a pattern that does not cover a portion ofthe elongated body 402. Alternatively, a portion of the elongated body402 can be masked prior to application of the insulating material.Removal of the mask, after insulating material application, can exposethe portion of the elongated body 402.

In some embodiments, the insulating material 404 comprises a polymer,for example, a non-conductive (that is, dielectric) polymer.Dip-coating, spray-coating, vapor-deposition, printing and/or other thinfilm and/or thick film coating or deposition techniques can be used todeposit the insulating material on the elongated body 402 and/or core410. For example, in some embodiments, the insulating material isapplied as a layer of from about less than 5 μm, or from about 5, about10 or about 15 μm to about 20, about 25, about 30, or about 35 μm ormore in thickness. The insulator can be applied as a single layer ofmaterial, or as two or more layers, which are comprised of either thesame or different materials, as described elsewhere herein.Alternatively, the conductive core does not require a coating ofinsulator. In some embodiments, the insulating material defines anelectroactive surface of the analyte sensor (that is, the workingelectrode). For example, a surface of the conductive core (such as, forexample, a portion of the first layer 412) can either remain exposedduring the insulator application, or a portion of applied insulator canbe removed to expose a portion of the conductive core's surface, asdescribed above.

In some embodiments, in which the sensor has an insulated elongated bodyor an insulator disposed upon a conductive structure, a portion of theinsulating material can be stripped or otherwise removed, for example,by hand, excimer lasing, chemical etching, laser ablation, grit-blasting(such as, for example, with sodium bicarbonate or other suitable grit),or the like, to expose the electroactive surfaces. In one exemplaryembodiment, grit blasting is implemented to expose the electroactivesurface(s), for example, by utilizing a grit material that issufficiently hard to ablate the polymer material yet also sufficientlysoft so as to minimize or avoid damage to the underlying metal electrode(for example, a platinum electrode). Although a variety of “grit”materials can be used (such as, for example, sand, talc, walnut shell,ground plastic, sea salt, and the like), in some embodiments, sodiumbicarbonate is an advantageous grit-material because it is sufficientlyhard to ablate, e.g., a parylene coating without damaging, e.g., anunderlying platinum conductor. An additional advantage of sodiumbicarbonate blasting includes its polishing action on the metal as itstrips the polymer layer, thereby eliminating a cleaning step that mightotherwise be necessary. Alternatively, a portion of an electrode orother conductive body can be masked prior to depositing the insulator inorder to maintain an exposed electroactive surface area.

The electroactive surface of the working electrode can be exposed byformation of a window 406 in the insulator 404. The electroactive window406 of the working electrode can be configured to measure theconcentration of an analyte.

In some embodiments, a silver wire is formed onto and/or fabricated intothe sensor and subsequently chloridized to form a silver/silver chloridereference electrode. Advantageously, chloridizing the silver wire asdescribed herein enables the manufacture of a reference electrode withgood in vivo performance. By controlling the quantity and amount ofchloridization of the silver to form silver/silver chloride, improvedbreak-in time, stability of the reference electrode and extended lifecan be obtained in some embodiments. Additionally, use of silverchloride as described above allows for relatively inexpensive and simplemanufacture of the reference electrode.

Referring to FIG. 3B and FIG. 3C, the reference electrode 414 cancomprise a silver-containing material (e.g., silver/silver chloride)applied over at least a portion of the insulating material 404, asdiscussed in greater detail elsewhere herein. For example, thesilver-containing material can be applied using thin film and/or thickfilm techniques, such as but not limited to dipping, spraying, printing,electro-depositing, vapor deposition, spin coating, and sputterdeposition, as described elsewhere herein. For example, a silver orsilver chloride-containing paint (or similar formulation) can be appliedto a reel of the insulated conductive core. Alternatively, the reel ofinsulated elongated body (or core) is cut into single unit pieces (thatis, “singularized”), and silver-containing ink is pad printed thereon.In still other embodiments, the silver-containing material is applied asa silver foil. For example, an adhesive can be applied to an insulatedelongated body, around which the silver foil can then be wrapped in.Alternatively, the sensor can be rolled in Ag/AgCl particles, such thata sufficient amount of silver sticks to and/or embeds into and/orotherwise adheres to the adhesive for the particles to function as thereference electrode. In some embodiments, the sensor's referenceelectrode includes a sufficient amount of chloridized silver that thesensor measures and/or detects the analyte for at least three days.

FIG. 2A is a cross-sectional view through a sensor illustrating oneembodiment of the membrane system 32. In this particular embodiment, themembrane system includes an electrode layer 42, an enzyme layer 44, adiffusion resistance layer 46, and a biointerface layer 48, all of whichare located around a working electrode of the sensor 38, and all ofwhich are described in more detail elsewhere herein. In someembodiments, a unitary diffusion resistance domain and biointerfacelayer can be included in the membrane system (e.g., wherein thefunctionality of both layers is incorporated into one domain). In someembodiments, the sensor is configured for short-term implantation (e.g.,from about 1 to 30 days). However, it is understood that the membranesystem 32 can be modified for use in other devices, for example, byincluding only one or more of the domains, or additional domains.

FIG. 2B is a cross-sectional view through one embodiment of the sensor,illustrating another embodiment of the membrane system 32. In thisparticular embodiment, the membrane system includes an interferencereduction or blocking layer 43, an enzyme layer 44, a diffusionresistance layer 46, and a biointerface layer 48 located around theworking electrode of a sensor 38, all of which are described in moredetail elsewhere herein.

FIG. 2C is a cross-sectional view through one embodiment of the sensor,illustrating still another embodiment of the membrane system 32. In thisparticular embodiment, the membrane system includes an interferentreduction or blocking layer 43, an enzyme layer 44, and a unitarydiffusion resistance/biointerface layer 47 located around the workingelectrode of a sensor, all of which are described in more detailelsewhere herein.

In some embodiments, the membrane system can include a biointerfacelayer 48, comprising a surface-modified biointerface polymer asdescribed in more detail elsewhere herein. However, the sensingmembranes 32 of some embodiments can also include a plurality of domainsor layers including, for example, an electrode domain (e.g., asillustrated in the FIG. 2A), an interference reduction or blockingdomain (e.g., as illustrated in FIGS. 2B and 2C), or a cell disruptivedomain (not shown), such as described in more detail elsewhere hereinand in U.S. Patent Publication No. US-2006-0036145-A1, which isincorporated herein by reference in its entirety.

It is to be understood that sensing membranes modified for othersensors, for example, can include fewer or additional layers. Forexample, in some embodiments, the membrane system can comprise oneelectrode layer, one enzyme layer, and two biointerface layers, but inother embodiments, the membrane system can comprise one electrode layer,two enzyme layers, and one biointerface layer. In some embodiments, thebiointerface layer can be configured to function as the diffusionresistance domain and control the flux of the analyte (e.g., glucose) tothe underlying membrane layers.

In some embodiments, one or more domains of the sensing membranes can beformed from materials such as silicone, polytetrafluoroethylene,polyethylene-co-tetrafluoroethylene, polyolefin, polyester,polycarbonate, biostable polytetrafluoroethylene, homopolymers,copolymers, terpolymers of polyurethanes, polypropylene (PP),polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT), polymethylmethacrylate (PMMA), polyether etherketone (PEEK), polyurethanes, polyurethane ureas, cellulosic polymers,poly(ethylene oxide), poly(propylene oxide) and copolymers and blendsthereof, polysulfones and block copolymers thereof including, forexample, di-block, tri-block, alternating, random and graft copolymers.

In some embodiments, the sensing membrane can be deposited on theelectroactive surfaces of the electrode material using known thin orthick film techniques (for example, spraying, electro-depositing,dipping, or the like). It should be appreciated that the sensingmembrane located over the working electrode does not have to have thesame structure as the sensing membrane located over the referenceelectrode; for example, the enzyme domain deposited over the workingelectrode does not necessarily need to be deposited over the referenceor counter electrodes.

Although the exemplary embodiments illustrated in FIGS. 2A through 2Cinvolve circumferentially extending membrane systems, the membranesdescribed herein can be applied to any planar or non-planar surface.

Sensor Electronics

In general, analyte sensor systems have electronics associatedtherewith, also referred to as a “computer system” that can includehardware, firmware, or software that enable measurement and processingof data associated with analyte levels in the host. In one exemplaryembodiment of an electrochemical sensor, the electronics include apotentiostat, a power source for providing power to the sensor, andother components useful for signal processing. In additionalembodiments, some or all of the electronics can be in wired or wirelesscommunication with the sensor or other portions of the electronics. Forexample, a potentiostat disposed on the device can be wired to theremaining electronics (e.g. a processor, a recorder, a transmitter, areceiver, etc.), which reside on the bedside. In another example, someportion of the electronics is wirelessly connected to another portion ofthe electronics (e.g., a receiver), such as by infrared (IR) orradiofrequency (RF). It is contemplated that other embodiments ofelectronics can be useful for providing sensor data output, such asthose described in U.S. Patent Publication No. US-2005-0192557-A1, U.S.Patent Publication No. US-2005-0245795-A1, U.S. Patent Publication No.US-2005-0245795-A1, U.S. Patent Publication No. US-2005-0245795-A1, U.S.Patent Publication No. US-2008-0119703-A1, and U.S. Patent PublicationNo. US-2008-0108942-A1, each of which is incorporated herein byreference in its entirety.

In one preferred embodiment, a potentiostat is operably connected to theelectrode(s) (such as described elsewhere herein), which biases thesensor to enable measurement of a current signal indicative of theanalyte concentration in the host (also referred to as the analogportion). In some embodiments, the potentiostat includes a resistor thattranslates the current into voltage. In some alternative embodiments, acurrent to frequency converter is provided that is configured tocontinuously integrate the measured current, for example, using a chargecounting device. In some embodiments, the electronics include an A/Dconverter that digitizes the analog signal into a digital signal, alsoreferred to as “counts” for processing. Accordingly, the resulting rawdata stream in counts, also referred to as raw sensor data, is directlyrelated to the current measured by the potentiostat.

In general, the electronics include a processor module that includes thecentral control unit that controls the processing of the sensor system.In some embodiments, the processor module includes a microprocessor,however a computer system other than a microprocessor can be used toprocess data as described herein, for example an ASIC can be used forsome or all of the sensor's central processing. The processor typicallyprovides semi-permanent storage of data, for example, storing data suchas sensor identifier (ID) and programming to process data streams (forexample, programming for data smoothing or replacement of signalartifacts such as is described in U.S. Patent Publication No.US-2005-0043598-A1). The processor additionally can be used for thesystem's cache memory, for example for temporarily storing recent sensordata. In some embodiments, the processor module comprises memory storagecomponents such as ROM, RAM, dynamic-RAM, static-RAM, non-static RAM,EEPROM, rewritable ROMs, flash memory, and the like.

In some embodiments, the processor module comprises a digital filter,for example, an infinite impulse response (IIR) or finite impulseresponse (FIR) filter, configured to smooth the raw data stream.Generally, digital filters are programmed to filter data sampled at apredetermined time interval (also referred to as a sample rate). In someembodiments, wherein the potentiostat is configured to measure theanalyte at discrete time intervals, these time intervals determine thesample rate of the digital filter. In some alternative embodiments,wherein the potentiostat is configured to continuously measure theanalyte, for example, using a current-to-frequency converter asdescribed above, the processor module can be programmed to request adigital value from the A/D converter at a predetermined time interval,also referred to as the acquisition time. In these alternativeembodiments, the values obtained by the processor are advantageouslyaveraged over the acquisition time due the continuity of the currentmeasurement. Accordingly, the acquisition time determines the samplerate of the digital filter.

In some embodiments, the processor module is configured to build thedata packet for transmission to an outside source, for example, an RFtransmission to a receiver. Generally, the data packet comprises aplurality of bits that can include a preamble, a unique identifieridentifying the electronics unit, the receiver, or both, (e.g., sensorID code), data (e.g. raw data, filtered data, or an integrated value) orerror detection or correction. Preferably, the data (transmission)packet has a length of from about 8 bits to about 128 bits, preferablyabout 48 bits; however, larger or smaller packets can be desirable incertain embodiments. The processor module can be configured to transmitany combination of raw or filtered data. In one exemplary embodiment,the transmission packet contains a fixed preamble, a unique ID of theelectronics unit, a single five-minute average (e.g. integrated) sensordata value, and a cyclic redundancy code (CRC).

In some embodiments, the processor further performs the processing, suchas storing data, analyzing data streams, calibrating analyte sensordata, estimating analyte values, comparing estimated analyte values withtime corresponding measured analyte values, analyzing a variation ofestimated analyte values, downloading data, and controlling the userinterface by providing analyte values, prompts, messages, warnings,alarms, and the like. In such cases, the processor includes hardware andsoftware that performs the processing described herein, for exampleflash memory provides permanent or semi-permanent storage of data,storing data such as sensor ID, receiver ID, and programming to processdata streams (e.g., programming for performing estimation and otheralgorithms described elsewhere herein) and random access memory (RAM)stores the system's cache memory and is helpful in data processing.Alternatively, some portion of the data processing (such as describedwith reference to the processor elsewhere herein) can be accomplished atanother (e.g., remote) processor and can be configured to be in wired orwireless connection therewith.

In some embodiments, an output module, which is integral with oroperatively connected with the processor, includes programming forgenerating output based on the data stream received from the sensorsystem and it's processing incurred in the processor. In someembodiments, output is generated via a user interface.

Interferents

Interferents are molecules or other species that can cause a sensor togenerate a false positive or negative analyte signal (e.g., anon-analyte-related signal). Some interferents become reduced oroxidized at the electrochemically reactive surfaces of the sensor, whileother interferents interfere with the ability of the enzyme (e.g.,glucose oxidase) used to react with the analyte being measured. Yetother interferents react with the enzyme (e.g., glucose oxidase) toproduce a by-product that is electrochemically active. Interferents canexaggerate or mask the response signal, thereby leading to false ormisleading results. For example, a false positive signal can cause thehost's analyte concentration (e.g., glucose concentration) to appearhigher than the true analyte concentration. False-positive signals canpose a clinically significant problem in some conventional sensors. Forexample in a severe hypoglycemic situation, in which the host hasingested an interferent (e.g., acetaminophen), the resultingartificially high glucose signal can lead the host to believe that he iseuglycemic or hyperglycemic. In response, the host can makeinappropriate treatment decisions, such as by injecting himself with toomuch insulin, or by taking no action, when the proper course of actionwould be to begin eating. In turn, this inappropriate action or inactioncan lead to a dangerous hypoglycemic episode for the host. Accordingly,certain embodiments contemplated herein include a membrane system thatsubstantially reduces or eliminates the effects of interferents onanalyte measurements. These membrane systems can include one or moredomains capable of blocking or substantially reducing the flow ofinterferents onto the electroactive surfaces of the electrode can reducenoise and improve sensor accuracy as described in more detail in U.S.Patent Publication No. US-2009-0247856-A1.

Drift

The term “drift” as used herein is a broad term, and is to be given itsordinary and customary meaning to a person of ordinary skill in the art(and is not to be limited to a special or customized meaning), andrefers without limitation to a change in the sensitivity of a sensorover time. Drift can be driven by a change in permeability of the sensormembrane system, which can be particularly evident in embodiments whichuse a polyurethane diffusion resistance domain. Without wishing to bebound by theory, it is believed that the change in permeability in suchsystems arises from the rearrangement of the diffusion resistance domainpolyurethane polymer chains to either bring more hydrophilic componentsto the surface or otherwise rearrange in some way to allow for greateraccess to hydrophilic polymer components during hydration of themembrane system. Because of this, increasing the speed of hydration orincreasing the wettability of the membrane system reduces system drift.

Due to electrostatically induced hydration, polymers and cross-linkedcoatings of zwitterionic compounds have near instantaneous wettingproperties. As discussed in greater detail below, including one or morezwitterionic compounds, precursors or derivatives thereof (such ashydrolyzable cationic esters) in the outermost domain of a membranesystem or applying a coating of such compounds to the surface of themembrane system results in reduced sensor drift. Further, as discussedin greater detail below, including a base polymer having a lowest glasstransition temperature as measured using ASTM D3418 of greater than −50°C. and an ultimate tensile strength as measured by ASTM D1708 that isgreater than 6000 psi can result in reduced sensor drift. The drift canbe characterized by less than 10% change in signal at 2 hrs after start.

Membrane Fabrication

Polymers of the preferred embodiments can be processed by solution-basedtechniques such as spraying, dipping, casting, electrospinning, vapordeposition, spin coating, coating, and the like. Water-based polymeremulsions can be fabricated to form membranes by methods similar tothose used for solvent-based materials. In both cases the evaporation ofa volatile liquid (e.g., organic solvent or water) leaves behind a filmof the polymer. Cross-linking of the deposited film or layer can beperformed through the use of multi-functional reactive ingredients by anumber of methods. The liquid system can cure by heat, moisture,high-energy radiation, ultraviolet light, or by completing the reaction,which produces the final polymer in a mold or on a substrate to becoated.

In some embodiments, the wetting property of the membrane (and byextension the extent of sensor drift exhibited by the sensor) can beadjusted and/or controlled by creating covalent cross-links betweensurface-active group-containing polymers, functional-group containingpolymers, polymers with zwitterionic groups (or precursors orderivatives thereof), and combinations thereof. Cross-linking can have asubstantial effect on film structure, which in turn can affect thefilm's surface wetting properties. Crosslinking can also affect thefilm's tensile strength, mechanical strength, water absorption rate andother properties.

Cross-linked polymers can have different cross-linking densities. Incertain embodiments, cross-linkers are used to promote cross-linkingbetween layers. In other embodiments, in replacement of (or in additionto) the cross-linking techniques described above, heat is used to formcross-linking. For example, in some embodiments, imide and amide bondscan be formed between two polymers as a result of high temperature. Insome embodiments, photo cross-linking is performed to form covalentbonds between the polycationic layers(s) and polyanionic layer(s). Onemajor advantage to photo-cross-linking is that it offers the possibilityof patterning. In certain embodiments, patterning using photo-crosslinking is performed to modify the film structure and thus to adjust thewetting property of the membrane.

Polymers with domains or segments that are functionalized to permitcross-linking can be made by methods known in the art. For example,polyurethaneurea polymers with aromatic or aliphatic segments havingelectrophilic functional groups (e.g., carbonyl, aldehyde, anhydride,ester, amide, isocyano, epoxy, allyl, or halo groups) can be crosslinkedwith a crosslinking agent that has multiple nucleophilic groups (e.g.,hydroxyl, amine, urea, urethane, or thio groups). In furtherembodiments, polyurethaneurea polymers having aromatic or aliphaticsegments having nucleophilic functional groups can be crosslinked with acrosslinking agent that has multiple electrophilic groups. Stillfurther, polyurethaneurea polymers having hydrophilic segments havingnucleophilic or electrophilic functional groups can be crosslinked witha crosslinking agent that has multiple electrophilic or nucleophilicgroups. Unsaturated functional groups on the polyurethane urea can alsobe used for crosslinking by reacting with multivalent free radicalagents. Non-limiting examples of suitable cross-linking agents includeisocyanate, carbodiimide, glutaraldehyde, aziridine, silane, or otheraldehydes, epoxy, acrylates, free-radical based agents, ethylene glycoldiglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE),or dicumyl peroxide (DCP). In one embodiment, from about 0.1% to about15% w/w of cross-linking agent is added relative to the total dryweights of cross-linking agent and polymers added when blending theingredients (in one example, about 1% to about 10%). During the curingprocess, substantially all of the cross-linking agent is believed toreact, leaving substantially no detectable unreacted cross-linking agentin the final film.

Polymers disclosed herein can be formulated into mixtures that can bedrawn into a film or applied to a surface using any method known in theart (e.g., spraying, painting, dip coating, vapor depositing, molding,3-D printing, lithographic techniques (e.g., photolithograph), micro-and nano-pipetting printing techniques, silk-screen printing, etc.). Themixture can then be cured under high temperature (e.g., 50-150° C.).Other suitable curing methods can include ultraviolet or gammaradiation, for example.

Biointerface Domain

The biointerface layer is the domain or layer of an implantable deviceconfigured to interface with (i.e., contact) a biological fluid whenimplanted in a host or connected to the host (e.g., via an intravascularaccess device providing extracorporeal access to a blood vessel). Whenpresent on an analyte sensor, e.g., a continuous analyte sensorimplanted into a host, the biointerface layer can increase sensorlongevity and decrease sensor inaccuracy by reducing thebiomaterial-associated inflammation response. The antifouling propertiesof the biointerface layer can inhibit the accumulation of cells,proteins, and other biological species on the sensor. In someembodiments, the biointerface domain may be formed of a biointerfacedomain described in U.S. Provisional Application No. 62/273,142, filedDec. 30, 2015, which is hereby incorporated by reference herein in itsentirety.

The biointerface layers disclosed herein can be mechanically robust,resist damage upon implantation, and withstand degradation during thesensor implantation. Further, the disclosed biointerface layers do notmaterially affect the response time of the sensor or the diffusionresistance layer's properties. Also, the disclosed biointerface layerscan have hydrophilic properties that can have large amounts of wateruptake, and fast water uptake and quick stabilization, so that sensorstart-up is not negatively affected. The disclosed biointerface layersare also permeable to analytes (e.g., glucose) but resist adsorption ofproteins.

Some embodiments described herein can include membranes that comprise abiointerface layer 48 (see FIGS. 2A and 2B).

Furthermore, the disclosed biointerface layer can be the host ofpharmaceutical or bioactive agent that upon release from thebiointerface layer to the local tissue can effectively reduce or delayinflammation. The anti-inflammatory agents can be steroidal ornon-steroidal drugs and can be the scavengers of reactive oxygen species(ROS). Suitable anti-inflammatory agents include but are not limited to,for example, nonsteroidal anti-inflammatory drugs (NSAIDS) such asacetometaphen, aminosalicylic acid, aspirin, celecoxib, cholinemagnesium trisalicylate, diclofenac potassium, diclofenac sodium,diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin,interleukin (IL)-10, IL-6 mutein, anti-IL-6 iNOS inhibitors (forexample, L-NAME or L-NMDA), Interferon, ketoprofen, ketorolac,leflunomide, melenamic acid, mycophenolic acid, mizoribine, nabumetone,naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate,sulindac, and tolmetin; and corticosteroids such as cortisone,hydrocortisone, methylprednisolone, prednisone, prednisolone,betamethesone, beclomethasone dipropionate, budesonide, dexamethasonesodium phosphate, flunisolide, fluticasone propionate, paclitaxel,tacrolimus, tranilast, triamcinolone acetonide, betamethasone,fluocinolone, fluocinonide, betamethasone dipropionate, betamethasonevalerate, desonide, desoximetasone, fluocinolone, triamcinolone,triamcinolone acetonide, clobetasol propionate, and dexamethasone.

In some embodiments, the biointerface layer can comprise a polymerdescribed as a bioprotective layer in US Patent Publication2014-0094671, which is incorporated by reference herein at least for itsteachings of bioprotective layers in sensor membranes.

While not wishing to be bound by theory, it is believed that zwitteriongroups in the biointerface layer can attract, retain, and order thestructure of water at the polymer-biologic interface, resulting inreversible adsorption, no or reduced protein denaturization, and no orreduced cell activation (see FIG. 12). Other possible mechanisms for theantifouling properties of the biointerface layer are significantswelling, which can fill in spaces at the site of implantation and actas buffering zone (see FIG. 16).

In further examples, the biointerface layer can comprise materials thatrepel the adhesion and/or absorption of carbohydrates. While not wishingto be bound by theory, the attachment or absorption of carbohydrates atthe sensor membrane may affect the operation of the sensor. Thus havingmaterials that repel the adhesion and/or absorption of carbohydrates canhelp alleviate this problem.

In further examples, Factor H can be covalently conjugated onto thesurface of the biointerface layer. Factor H is one of the principalregulators of a complement system that play a vital role in the immuneresponse. Its regular functions include controlling proteins thatgenerate proinflammatory anaphylatoxins; maintaining tissue integrity byidentifying “self” from “non-self” and harnessing directanti-inflammatory properties. Thus, Factor H is an important proteinthat regulates complement activation. This regulation occurs by multiplemechanisms which include disruption of C3 convertase formation andacceleration of its decay. Factor H also acts as a cofactor to factor Iin the degradation of C3b, and competes with factor B for binding toC3b.

Factor H for use as a medical device surface coating can be beneficialbecause of its link between complement-triggered inflammations, theregulatory role of Factor H in stopping complement triggeredinflammation and tissue damage.

In this embodiment, Factor H can be covalently conjugated onto thesurface of biointerface layer by methods disclosed herein. Factor H canthen be released upon environmental changes and control inflammation.Control of inflammation can help reduce issues associated with first daysignal reduction or loss after the sensor is implanted. Factor H canalso help increase the longevity and decrease in vivo variability.

Thus, disclosed herein in a certain embodiment is a sensor comprising abiointerface layer comprising a covalently connected active Factor H tothe surface of the biointerface layer. The covalent attachment can be alinker, e.g., an alkyl, alkoxyl, ester, triazole, polyether, polyester,polyalkeneoxide, and the like. In some examples, the linker can besensitive to cleavage by internal or external stimuli, such as pH, heat,UV-Vis, or protease attack. The linker can also be an oligo peptidesequence which can be cleaved by MMP (Matrix metallopeptidase), whichare upregulated in atherosclerosis and inflammation. A schematic of abiointerface layer with conjugated Factor H is shown in FIG. 33. Thelayer comprises three parts: (i) the biointerface polymer as disclosedherein; (ii) stimuli responsive linker, which can be cleaved by changingof environment; and (iii) active Factor H, which is covalently connectedto linker.

The biointerface layer comprises a biointerface polymer. In someembodiments, the biointerface polymer is a polyzwitterion.Polyzwitterions are polymers where a repeating unit of the polymer chainis a zwitterionic moiety. As such, these polymers have the same numberof cationic and anionic groups, due to each zwitterion repeating unithaving both a positive and negative charge, and thus have an overallcharge of zero, often through a wide pH range.

Polyzwitterions are distinguishable from other polyampholytes in that,which polyampholytes contain anionic and cationic groups, the ionicgroups are not correlated with one another as part of the same repeatingunit. So the anionic and cationic groups may be distributed apart fromone another, at random intervals, or one ionic group may outnumber theother. It is thus typical for a polyampholyte to have a net charge,except perhaps at some narrow pH range.

The disclosed polyzwitterions can have a variety of repeating units,which are illustrated as i) through vii) below, where n is some integerfrom 2 to 1000:

In structures i) through iv) the zwitterionic unit is connected to thebackbone chain (

) and the charges are on side-groups that are pendant to the chain. Instructures v) through vii) the zwitterionic unit is such that one orboth charges is on the chain itself.

Examples of suitable zwitterionic monomers that can be used to produce apolyzwitterion of any of structures i) through vii) include:

-   -   ammoniophosphates (phosphobetaines or lecithin analogues),        ammoniophosphonates (phosphonobetaines), or ammoniophosphinates        (phosphinobetaines), respectively having the structures

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R², R³, and R⁴ are independently chosen from        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; wherein one or more of R¹, R², R³, R⁴, and Z are        substituted with a polymerization group;    -   ammoniosulfonates (sulfobetaines), ammoniosulfates, respectively        having the structures:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³, are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R′, R², R³, and Z are substituted with a        polymerization group; and    -   ammoniocarboxylates having the structures:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³ are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In each of these monomers Z can have a length of from 1 to 12 atoms,e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 atoms, where any ofthese values can form an upper or lower endpoint of a range.

In a step-growth polymerization, a matching pair of functional groupsare selected to promote polymerization, for example, to polymerize azwitterionic monomer bearing di-hydroxyl group, a diisocyanate, epoxide,or di-carboxylic acid group containing co-monomer can be chosen toafford polymers formed with urethane, ether, and ester linkages.

Additional examples of zwitterion precursors that be modified and formedinto monomers for the disclosed biointerface polymers includeammoniocarboxylate (caboxylbetaine) or ammoniosulfonates (sulfobetaine),phosphobetaines (ammoniophosphates or lecithin analogues),phosphatidylcholine, poly(carboxybetaine), poly(sulfobetaine), andprecursors or derivatives; trigonelline, ectoine,3-dimethylsulfoniopropanoate, arsenobetaine, ammoniophosphonates(phosphonobetaines), ammoniophosphinates(phosphinobetaines),ammoniosulfonamides, ammoni-sulfon-imides, guanidiniocarboxylates(asparagine analogs), pyridiniocarboxylates,ammonio(alokoxy)dicyanoethenolates, ammonioboronates,sulfoniocarboxylates, phosphoniosulfonates, phosphoniocarboxylates,squaraine dyes, and oxypyridine betaines.

These monomers can be prepared by methods known to those of skilled inthe art, e.g., as detailed in Laschewsky, “Structures and synthesis ofzwitterionic polymers,” Polymers 6:1544-1601, 2014. In certain examples,the disclosed polyzwitterions can have repeating zwitterionic unitsobtained from any of the zwitterionic monomers disclosed above.

The biointerface polymer may also comprises polyurethane and/or polyureasegments. For example, the biointerface polymer can comprise apolyurethane copolymer such as polyether-urethane-urea,polycarbonate-urethane, polyether-urethane, silicone-polyether-urethane,silicone-polycarbonate-urethane, polyester-urethane, polyurethane-urea,and the like. Since these polyurethane and/or polyurea segments containurea and/or urethane bonds formed from polyisocyanate and short chainpolyol or polyamine, which are hydrogen bonding rich moieties, thesesegments are referred to herein as “hard segments.” These segments canalso be relatively hydrophobic.

In addition to polyurethane and/or polyurea hard segments, the disclosedbiointerface polymers can also comprise soft segments, which haverelatively poor hydrogen bonding. Soft segment are usually composed ofpolyols of polycarbonates, polyesters, polyethers, polyarylene, andpolyalkylene, and the like. The soft segments can be either hydrophobicor hydrophilic.

Although the biointerface polymer in some embodiments comprisespolyurethane and/or polyurea, in other embodiments, the biointerfacepolymer may be a polymer that does not comprise polyurethane and/orpolyurea.

Biointerface polymers useful for certain embodiments can include linearor branched polymer on the backbone structure of the polymer. Thus,either the hard or soft segments can contain branching or linearbackbones.

The zwitterionic monomers can be part of either the hard or softsegments, or both, as described herein.

In some embodiments, the hard segment portion of the biointerfacepolymer can comprise from about 5% to about 50% by weight of thepolymer, sometimes from about 15% to 20%, and other times from about 25%to 40%. The hard segments can have a molecular weight of from about 160daltons to about 10,000 daltons, and sometimes from about 200 daltons toabout 2,000 daltons. In some embodiments, the molecular weight of thesoft segments can be from about 200 daltons to about 10,000,000 daltons,and sometimes from about 500 daltons to about 5,000 daltons, andsometimes from about 500 daltons to about 2,000 daltons.

As noted the hard segments can be polyurethanes or polyureas.Polyurethane is a polymer produced by the condensation reaction of adiisocyanate and a difunctional hydroxyl-containing material. A polyureais a polymer produced by the condensation reaction of a diisocyanate anda difunctional amine-containing material. Preferred diisocyanatesinclude aliphatic diisocyanates containing from about 4 to about 9methylene units. Diisocyanates containing cycloaliphatic moieties canalso be useful in the preparation of the polymer and copolymercomponents of the membranes of preferred embodiments.

The soft segments used in the preparation of the biointerface polymercan be a polyfunctional aliphatic polyol, a polyfunctional aliphatic oraromatic amine, or the like that can be useful for creating permeabilityof the analyte (e.g., glucose) therethrough, and can include, forexample, polyoxazoline, poly(ethylene glycol) (PEG), polyacrylamide,polyimine, polypropylene oxide (PPO), PEG-co-PPO diol, silicone-co-PEGdiol, Silicone-co-PPO diol, polyethylacrylate (PEA),polyvinylpyrrolidone (PVP), and variations thereof (e.g., PVP vinylacetate), and wherein PEG and variations thereof can be preferred fortheir hydrophilicity.

In some of the embodiments, the soft segment portion of the biointerfacepolymer can comprise from about 5% to about 50% by weight of thepolymer, sometimes from about 15% to 20%, and other times from about 25%to 40%. The soft segments can have a molecular weight of from about 160daltons to about 10,000 daltons, and sometimes from about 200 daltons toabout 2,000 daltons. In some embodiments, the molecular weight of thesoft segments can be from about 200 daltons to about 10,000,000 daltons,and sometimes from about 500 daltons to about 5,000 daltons, andsometimes from about 500 daltons to about 2,000 daltons.

In some embodiments, the biointerface polymer, including hard and softsegments, and zwitterionic repeating units, can have a molecular weightof from about 10 kDa to about 500,000 kDa, for example, from about 10kDa to about 100,000 kDa, from about 1000 kDa to about 500,000 kDa, fromabout 10,000 kDa to about 100,000 kDa, and from about 100,000 kDa toabout 500,000 kDa.

The hard and soft segments can each be selected for their properties,such as, but not limited to, tensile strength, flex life, modulus, andthe like. For example, polyurethanes are relatively strong and providenumerous reactive pathways, which properties can be advantageous as bulkproperties for a membrane domain of the continuous sensor.

In some specific examples, the segments can be chosen to result in abiointerface polymer with high Tg. Having a high Tg segment or polymerin the biointerface layer can result in stronger mechanical properties.Further, a hig Tg segment or polymer can allow for more hydrophilic softsegments, which can allow for the incorporation of bioactive agents likeanti-inflammatory drugs (e.g., dexamethasone). As an example, thezwitterionic feature of the betaine can bind salt form of dexamethasonemore efficiently because of the electron static interactions. As such,disclosed herein are examples of biointerface polymers wherein thehydrophobic segments can be composed of high glass transitiontemperature (T_(g)) hydrophilic polymers (e.g., polycarbonates). Thehigh T_(g) hydrophobic segments can have strong mechanical propertiesand thus construct the porous scaffold which could absorb andencapsulate water and drug molecules inside for continuous drugdelivery. The materials can also covalently incorporate functionalreactive groups for further chemical reaction or crosslinking (thosegroups include carboxylic acid, azide, alkyne, alkene, thiol).

As noted, the biointerface polymer contains one or more zwitterionicrepeating units; thus these groups are “internal” in reference to thepolymer backbone. Such “internal” repeating units are distinguished froma material that is found at the end of a polymer chain since such amoiety would only be bonded to the polymer chain at one location. Thedisclosed biointerface polymers can, in some embodiments, have one ormore zwitterionic groups at the terminal ends of the polymer chains;however, such groups are not the only zwitterionic groups in the chain;there is at least one internal zwitterionic group in the backbone.

In some preferred embodiments, zwitterion moieties are selected fordesirable properties, for example, non-constant noise-blocking ability,break-in time (reduced), ability to repel charged species, cationic oranionic blocking, surface wettability, antifouling, or the like. In someembodiments, the zwitterion or zwitterion precursor exists aszwitterionic groups while the device is in vivo. As such, these groupspresent mixed charged areas of the device surface to the surroundingenvironment, thereby increasing surface hydration of the device, andpotentially reducing nonspecific protein adsorption and cell adhesion.

In some embodiments, the biointerface polymer includes at least about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,about 28%, about 29%, about 30%, to about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54% or about 55% zwitterionic repeating units by weight ofthe polymer.

The zwitterionic repeating unit can be a betaine such as a carboxyl,sulfo, or phosphor betaine compound, precursor or derivative thereof(for example alkylbetaines or aminobetaines). These segments or moietiescan be incorporated into the biointerface polymer, whether in the hardsegment, the soft segment, or both, for example up to about 55 wt. % ofthe biointerface polymer.

Although in some embodiments, using two or more different zwitterion orzwitterion precursor segments or moieties are used, in otherembodiments, a single zwitterion or zwitterion precursor segment ormoiety can be used in the biointerface polymer.

Some examples of a biointerface polymer are schematically illustrated inFIG. 4. Generally, the biointerface polymer comprises one or more hardsegments and one or more soft segments. The hard segments can bealiphatic or aromatic monomers. The soft segments can be hydrophilic orhydrophobic oligomers of, for example, polyalkylene glycols,polycarbonates, polyesters, polyethers, polyvinylalcohol,polyvinypyrrolidone, polyoxazoline, and the like. The zwitterionicgroups (e.g., betaines) can be part of the soft segment, the hardsegments, or both. As illustrated, in FIG. 4, various hard and softsegments can be present, which permits one to tune the properties of thebiointerface polymer by using different segments, different segmentslengths, functionalization on certain segments, crosslinking certainsegments, and the like. In some embodiments, biocompatible segmentedblock polyurethane copolymers comprising hard and soft segments can beused for the biointerface layer.

Incorporation of these zwitterionic repeating units into a polymer canbe achieved by using zwitterionic monomers that have diols or diamines(e.g., at position Z), or can be attached to diols or diamines at any ofR¹ through R⁴. Attaching a diol or diamine at R¹-R⁴ can be accomplishedby reacting the corresponding precursor with a halo-substituted diamineor halo-substituted diol. Examples of such monomers are shown below:

-   -   where W, Y, and Z are, independently, branched or straight chain        alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or        heteroaryl, any of which can be optionally substituted with O,        OH, halogen, amido, or alkoxyl; R¹ is H, alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; and R², R³,        and R⁴, are independently chosen from alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl. In specific        examples W is C₁-C₄ alkyl. In specific examples Y is C₁-C₄        alkyl. In other examples Z is C₁-C₄ alkyl.

These compounds can be reacted with a diisocyanate to form apolyurethane or polyurea. Alternatively, the carboxylates, sulfonates,phosphinates, or phophonates moieties can be protected and then theprotection group can be removed after polymerization. In anotheralternative, the amine can be a tertiary amine, which is thenquaternized by alkylation after polymerization.

Another method involves the radical polymerization of zwitterionicmonomers having unsaturated moieties substituted at position Z in themonomers shown above. In other examples zwitterionic monomers where anunsaturated moiety is attached to the ammonium group can be used in aradical polymerization. Examples of such monomers are shown below:

-   -   where X is O, NH, or NR⁴, Y and Z are, independently, branched        or straight chain alkyl, heteroalkyl, cycloalkyl,        cycloheteroalkyl, aryl, or heteroaryl, and of which can be        optionally substituted with OH, halogen, or alkoxyl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or        heteroaryl; and R³ and R⁵ are independently chosen from        heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl.        In specific examples, R⁵ is H or CH₃. In other examples, X is O.        In still other examples, X is NH or NCH₃. In specific examples Y        is C₁-C₄ alkyl. In other examples Z is C₁-C₄ alkyl.

Additional examples of suitable zwitterionic monomers includeN-(2-methacryloyloxy)ethyl-N,N-dimethylammonio propanesulfonate,N-(3-methacryloylimino)propyl-N,N-dimethylammonio propanesulfonate,2-(methacryloyloxy)ethylphosphatidylcholine, and3-(2′-vinyl-pyridinio)propanesulfonate.

In other embodiments, the biointerface polymer is crosslinked. Forexample, polyurethaneurea polymers with aromatic or aliphatic segmentshaving electrophilic functional groups (e.g., carbonyl, aldehyde,anhydride, ester, amide, isocyanate, epoxy, allyl, or halo groups) canbe crosslinked with a crosslinking agent that has multiple nucleophilicgroups (e.g., hydroxyl, amine, urea, urethane, or thio groups). Infurther embodiments, polyurethaneurea polymers having aromatic oraliphatic segments having nucleophilic functional groups can becrosslinked with a crosslinking agent that has multiple electrophilicgroups. Still further, polyurethaneurea polymers having hydrophilicsegments having nucleophilic or electrophilic functional groups can becrosslinked with a crosslinking agent that has multiple electrophilic ornucleophilic groups. Unsaturated functional groups on the polyurethaneurea can also be used for crosslinking by reacting with multivalent freeradical agents.

Non-limiting examples of suitable cross-linking agents includeisocyanate, carbodiimide, gluteraldehyde or other aldehydes, aziridine,silane, epoxy, acrylates, free-radical based agents, ethylene glycoldiglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE),or dicumyl peroxide (DCP). In one embodiment, from about 0.1% to about15% w/w of cross-linking agent is added relative to the total dryweights of cross-linking agent and polymers added when blending theingredients (in one example, about 1% to about 10%). During the curingprocess, substantially all of the cross-linking agent is believed toreact, leaving substantially no detectable unreacted cross-linking agentin the final layer.

Further, the disclosed biointerface layer can have zwitterions entrappedor embedded within the polymer network by non-covalent interactions.Thus, in further embodiments, the disclosed biointerface layer cancomprise a biointerface polymer and additional betaines blendedtherewith. For example, the biointerface polymer can be blended withcocamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine,caprylyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine,palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine),octyl betaine, phosphatidylcholine, glycine betaine,poly(carboxybetaine) (pCB), and poly(sulfobetaine) (pSB). It will beappreciated that many more zwitterionic compounds or precursors orderivatives thereof can be applicable and that this list of exemplarybetaines is not intended to limit the scope of the embodiments.

The biointerface layer can further comprise a biointerface domain,wherein the biointerface domain comprises a surface modifying polymeradded to a base polymer, wherein the surface modifying polymer comprisesa polymer chain having both hydrophilic and hydrophobic regions andwherein one or more zwitterionic compounds are covalently bonded to aninternal region of the polymer, wherein the base polymer can be selectedfrom silicone, epoxies, polyolefins, polystylene, polyoxymethylene,polysiloxanes, polyethers, polyacrylics, polymethacrylic, polyesters,polycarbonates, polyamide, poly(ether ketone), poly(ether imide),polyurethane, and polyurethane urea.

In some embodiments, the biointerface layer can comprise a combinationof one or more biointerface polymer(s), for example, polyurethane orpolyurethane urea and one or more hydrophilic polymers, such as, PVA,PEG, polyacrylamide, polyacetates, polyzwitterions, PEO, PEA, PVP, andvariations thereof (e.g., PVP vinyl acetate), e.g., as a physical blendor admixture wherein each polymer maintains its unique chemical nature.

In some embodiments, the biointerface layer 48 is positioned mostdistally to the sensing region such that its outer most domain contactsa biological fluid when inserted in vivo. In some embodiments, thebiointerface layer is resistant to cellular attachment, impermeable tocells, and can be composed of a biostable material. While not wishing tobe bound by theory, it is believed that when the biointerface domain 48is resistant to cellular attachment (for example, attachment byinflammatory cells, such as macrophages, which are therefore kept asufficient distance from other domains, for example, the enzyme domain),hypochlorite and other oxidizing species are short-lived chemicalspecies in vivo and biodegradation does not generally occur.Additionally, the materials preferred for forming the biointerfacedomain 48 can be resistant to the effects of these oxidative species andhave thus been termed biodurable. In some embodiments, the biointerfacedomain controls the flux of oxygen and other analytes (for example,glucose) to the underlying enzyme domain (e.g. wherein the functionalityof the diffusion resistance domain is built-into the biointerface domainsuch that a separate diffusion resistance domain is not required).

In some embodiments, the one or more zwitterionic compounds orprecursors thereof applied to the surface of the membrane system arehydrolyzable cationic esters of zwitterionic compounds. In theseembodiments, the hydrolyzable cationic esters provide the added benefitthat hydrolysis of the cationic esters into nonfouling zwitterionicgroups can kill microbes (such as bacteria) or condense DNA. Further,the mixed-charge nature of the resulting zwitterionic groups result ininhibition of nonspecific protein adsorption on the surface of thesensors. In these embodiments, cationic betaine esters, such as cationicpCB esters are preferable.

In certain embodiments, the biointerface polymer can comprise reactivegroups that can be available for further functionalization. For example,unsaturated functional groups like alkynes can be used to attach variousmoieties attached to dipolar groups likes azides to form covalentlinkages. Such Huisgen cycloaddition chemistry is often referred to asclick chemistry. Thus, in certain embodiments herein the biointerfacelayer can comprise alkyne functional groups pendant on the polymerbackbone. Antifouling agents such as proteins, cytokines,anti-inflammatory agent, steroids, and other bioactive agents disclosedherein, which are attached to a dipolar group like an azide, can beconveniently attached to the polymer, resulting in a triazole group.Thus, disclosed herein are biointerface layers, sensors containing suchlayers, that comprise an alkyne, triazole, or both. These reactivegroups can be present in the zwitterion repeating units (e.g., assubstituents on Z or Y). A schematic of these products is shown in FIG.17.

It has been found that incorporation of zwitterion or zwitterionprecursor segments or moieties internally in the polymer backbone can bechallenging due to the solubility issues associated with the monomers ofthe zwitterion or zwitterion precursors. Such groups typically can onlybe dissolved in highly polar solvents such as methanol and water, whichare not favorable in the synthesis of some the biointerface polymers(e.g., polyurethanes). Thus, the available functional groups that couldbe used chemically incorporated into the biointerface polymer's backboneby solution based polycondensation synthesis was limited. As analternative method of incorporating zwitterion or zwitterion precursorsegments or moieties into the base-polymer's backbone, precursors orderivatives of the zwitterion or zwitterion precursors can be used. Forexample, zwitterion precursors and/or zwitterionic derivatives, whichhave more desirable solubility characteristics in low polarity organicsolvents, can be used as monomers. The biointerface polymer (e.g.,polyurethaneureas) can be synthesized by polycondensation reactions andform well-defined polymers with high molecular weight and lowpolydispersity index. These polymers can then be converted to zwitteriongroup containing polymers via chemical reaction (such as hydrolysis,deprotection, heat-triggered rearrangement, and UV-triggereddegradation) or biological triggered reaction after in vivo implantationof the device.

In some embodiments, the hydrophilic segment of the biointerface domaincomprises a “brush” polymer where a linear polymer backbone isfunctionalized with oligomers of hydrophilic branches (e.g., PEG). Thatis, in the biointerface domain, the biointerface polymer can comprises apolymer chain having both hydrophilic and hydrophobic regions and thehydrophilic regions can comprise a liner polymer chain havinghydrophilic oligomers bound thereto and the linear polymer can begrafted to the biointerface polymer. The liner polymer can be anon-biodegradable polymer, e.g., polyacrylate, that is functionalized atone end (e.g., azide) to permit attachment to functional groups locatedon main chain of the biointerface domain. For example, a coppercatalyzed azide-alkyne Huisgen cycloaddition reaction (CuAAC) can beused to attach multiple brush polymers to alkyne functional groups onthe biointerface domain. The brush polymers can be prepared from AtomTransfer Radical Polymerization from homopolymers with a defined chainlength (e.g., PEG-acrylate monomers). In some embodiments, thebiointerface layer is grafted onto surface functional groups in anadjacent layer (e.g., resistance layer) (see e.g., FIG. 14).

In another embodiment, the biointerface layer can comprise anamphiphilic copolymer of a hydrophobic, hyperbranched fluoropolymer(HBFP) and hydrophilic polymer such as polyalkyloxide, polyvinylalcohol,or polyester (see e.g., FIG. 15). These networks can be prepared fromhyperbranched fluoropolymer (Mn from 1 to 100 kDa, e.g., from 5 to 15kDa), by atom transfer radical-self condensing vinyl copolymerizationand linear diamine-terminated hydrophilic polymer such asdiamino-poly(ethylene glycol) (Mn from 1 to 20,000 Da). Thus, disclosedherein is a continuous analyte sensor comprising a amphiphilic copolymercomprising hyperbranched, fluoropolymer segments and hydrophilicpolyethyleneglycol segments. Examples of such polymers are disclosed inGudipati et al. J. Polymer Sci. (42:6193-6208, (2004); and Muller etal., Macromolecules 31:776, (1998), which are incorporated by referenceherein for their teachings of amphiphilic polymers comprising HBFP.

In another embodiment, a fluorescent dye (e.g., Rhodamine) can becovalently incorporated into the biointerface domain. This domain canpermit the tracking of the domain, sensing layer, and/or sensor withconfocal microscopy. This feature can aid in tracking the degradation orphase separation of the polymers in the sensing membrane. Such polymerscan be prepared by a two-step polycondensation. It certain embodiments,the fluorescent dye incorporated biointerface domains can be combinedand blended with other biointerface domains as disclosed herein.Examples of suitable fluorescent dyes include, but are not limited to,benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine;polycyclic aromatic hydrocarbons such as perylene, perylene diimine,pyrenes; azo dyes; xanthene dyes; boron dipyoromethene, aza-borondipyoromethene, cyanine dyes, metal-ligand complex such as bipyridine,bipyridyls, phenanthroline, coumarin, and acetylacetonates of rutheniumand iridium; acridine, oxazine derivatives such as benzophenoxazine;aza-annulene, squaraine; 8-hydroxyquinoline, polymethines, luminescentproducing nanoparticle, such as quantum dots, nanocrystals; carbostyril;terbium complex; inorganic phosphor; ionophore such as crown ethersaffiliated or derivatized dyes; or combinations thereof. Specificexamples of suitable fluorescent dyes include, but are not limited to,Pd (II) octaethylporphyrin; Pt (II)-octaethylporphyrin; Pd (II)tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II)meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetraphenymetrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II)octaethylporphyrin ketone; Pd (II)meso-tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra(pentafluorophenyl) porphyrin; Ru (II)tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)₃); Ru (II)tris(1,10-phenanthroline) (Ru(phen)₃), tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)₃); erythrosine B; fluorescein; eosin;iridium (III) ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin));indium (III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate);Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow;Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymolblue; xylenol blue; cresol red; chlorophenol blue; bromocresol green;bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7;4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein;chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red;nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein;eosin; 2′,7′-dichlorofluorescein; 5(6)-carboxy-fluorescein;carboxynaphtofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid;semi-naphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride;(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron;platinum (II) octaethylporphyin; dialkylcarbocyanine; anddioctadecylcycloxacarbocyanine; derivatives or combinations thereof.

The fluorescent labeled biointerface domain can contain from about 0.05wt. % to about 20 wt. % fluorescence dye, for example, from about 0.1,about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 15, or about 20 wt. %, where any ofthe stated values can form an upper or lower endpoint of a range.

In certain embodiments, the thickness of the biointerface domain can befrom about 0.1, about 0.5, about 1, about 2, about 4, about 6, about 8μm or less to about 10, about 15, about 20, about 30, about 40, about50, about 75, about 100, about 125, about 150, about 175, about 200 orabout 250 μm or more. In some of these embodiments, the thickness of thebiointerface domain can be sometimes from about 1 to about 5 μm, andsometimes from about 2 to about 7 μm. In other embodiments, thebiointerface domain can be from about 20 or about 25 μm to about 50,about 55, or about 60 μm thick. In some embodiments, the glucose sensorcan be configured for transcutaneous or short-term subcutaneousimplantation, and can have a thickness from about 0.5 μm to about 8 μm,and sometimes from about 4 μm to about 6 μm. In one glucose sensorconfigured for fluid communication with a host's circulatory system, thethickness can be from about 1.5 μm to about 25 μm, and sometimes fromabout 3 to about 15 μm. It is also contemplated that in someembodiments, the biointerface layer or any other layer of the electrodecan have a thickness that is consistent, but in other embodiments, thethickness can vary. For example, in some embodiments, the thickness ofthe biointerface layer can vary along the longitudinal axis of theelectrode end.

The biointerface layer can be hydrophilic as measured by contact angle.For example, the biointerface layer can have a contact angle of fromabout 20° to about 90°, from about 60 to about 90°, from about 70 toabout 90°, from about 80 to about 90°, from about 60 to about 80°, atleast about 50°, at least about 60°, or at least about 70°.

The biointerface layer can also have a low polydispersity index. Forexample, the polymer can have a polydispersity index of from about 1.4to about 3.5, from about 1.75 to about 2.25, from about 1.75 to about2.5, or about 2. The biointerface layer can also not materially affectthe T95 response time of a sensor. T95 response time is the amount oftime required for the electrical response to reach 95% of the differencebetween a 1^(st) and 2^(nd) glucose step's response. For example, asensor with a biointerface layer as disclosed herein can have a T95response time that is the same, or within 5% of, the T95 response timeof a sensor that is otherwise identical but without the biointerfacelayer.

Diffusion Resistance Domain

One shortcoming of some current diffusion-resistance layers, also calledthe dufussion resistance domain, is that they tend to cause significantsensor drift within a few days of the sensor usage, as indicated by bothin-vitro testing and in-vivo data. While not wishing to be bound bytheory, sensitivity is related, in part, to hydrophilic polymers in thediffusion-resistance layer. If the hydrophilic polymer leaches out overtime from the layer when under hydrated conditions, changes insensitivity and decreases in accuracy and longevity can occur.

Further, un-equilibrated phase separation among the two polymers canresult in unpredictable sensitivity changes over time and can requireextra calibrations during sensor use. An equilibrated and reproduciblephase separation between the hydrophilic and hydrophobic polymers canimprove senor accuracy, better consistency and reproducibility andsensor longevity. In some embodiments, the diffusion resistance domainmay be formed of a diffusion resistance domain described in U.S.Provisional Application No. 62/273,219, filed Dec. 30, 2015, which ishereby incorporated by reference herein in its entirety.

The diffusion resistance membrane also requires strong mechanicalstability to resist rubbing against tissue in vivo and keep the phaseseparated microstructure unchanging. Strong mechanical resistance layercan also minimize the membrane rearrangement, improve under layerstability (due to increased hoop strength), and reduce tip breachfailure (due to improved puncture resistance).

Disclosed herein is a stable, semipermeable diffusion-resistance layer.The disclosed diffusion-resistance layer comprises a base polymercapable of providing a structurally stable matrix. The discloseddiffusion-resistance layer also contains a hydrophilic analyte permeablephase that resides throughout the layer. The layer has amicrophase-separated morphology where the analyte permeable phases ispercolated throughout a main, hydrophobic polymer matrix. The mainmatrix polymer provides structural support as required by the membraneapplication as well as influence on the analyte permeable phaseseparation, the size and distribution of its microstructure. It is thesize and distribution of analyte permeable phase that determine thepermeability, hence the sensitivity of a given electrochemical sensor.It is therefore understandable and desirable to maintain the size anddistribution of analyte permeable phase upon initial deployment tominimize the sensitivity change over time of actual use and resisttemperature and mechanical stress fluctuation.

In specific embodiments, the disclosed diffusion-resistance layercomprises a polymer blend composed of a base polymer, which is ahydrophobic segmented block copolymer, and hydrophilic polymer. Thehydrophobic segmented block copolymer has a high glass transitiontemperature (T_(g)) to afford increase dimensional stability andresistance to swelling caused by hydration, temperature and mechanicalchallenges. By using a polymer with a high T_(g), a more rigid matrixcan be formed, which can limit the swelling induced by the hydration andmaintain the size and distribution of hydrophilic analyte permeablemicrophase.

At low temperature polymers are brittle, glassy since there is nosufficient energy present to encourage local or segmental chainmovement. As the temperature is increased and at some point there issufficient energy available to allow some chain mobility. For a polymercontaining both amorphous and crystalline phases or is only amorphous,the onset of this segmental chain mobility for the amorphous segments iscalled the glass transition temperature, T_(g). Because there isunoccupied volume in the amorphous polymer phase some segmental chainmovement occurs. This segmental chain movement is sometimes depicted asa snake slithering “in place” within the glass. The localized chainmovement causes a further increase in unoccupied volume, and largersegments are able to move eventually, allowing the snake furthermovement in the glass. Further increase in temperature can lead to theenergy sufficient to break up the crystalline phase, and thistemperature is often referred to as the melt transition temperature,T_(m). In gas and liquid separation membrane, mass transfer occurs inthe amorphous phase.

The flexibility of amorphous polymers is reduced drastically when theyare cooled below a characteristic transition temperature (T_(g)). Attemperature below T_(g) there is no ready segmental motion and as such apolymer with desirable glass transition temperature be selected forapplication that restrict the segmental mobility and afford structuraland dimensional stability. As a property associated with the polymer,T_(g) values are most often related to the onset of segmental motion inthe principle polymer backbone, the more rigid and bulky is the polymersegment the high the T_(g) is. In a multi-block urethane copolymer,multiple T_(g)'s associated with different soft-segments can present,for example, an extremely low T_(g) of −120° C., which can occur ifsilicone is used as co-soft segment. Likewise, a relatively high T_(g)of −50° C. or higher can be related to a more rigid polycarbonateco-soft-segment.

The glass transition temperature of the hydrophobic segmented blockcopolymer component of the diffusion resistance layer can be measuredusing ASTM D3418. In specific examples the lowest glass transitiontemperature of the hydrophobic segmented block copolymer can be greaterthan −50° C., e.g., greater than −40, greater than −30, greater than−20, greater than −10, or greater than 0° C. In other examples, thehydrophobic segmented block copolymer can have a lowest glass transitiontemperature of from 0° C. to −50° C., from −10° C. to −50° C., from −20°C. to −50° C., from 0 to −40° C., from −10° C. to −40° C., from −20° C.to −40° C., from 0° C. to −30° C., from −10° C. to −30° C., or from 0°C. to −30° C. A copolymer can have T_(g)'s associated with differentsegments. Thus, when there are multiple T_(g)'s, the Tg's referencedhere refer to the lowest T_(g) the copolymer has. As illustrated in FIG.43, the T_(g) of polyurethane block copolymer can be altered by changingthe amount and type of soft-segment incorporated in polyurethaneblock-copolymer synthesis. The silicone containing polyurethane exhibitsa low T_(g) at around −120° C. associated with silicone and is absentfrom the silicone-free polycarbonate urethane. The T_(g) can be furtherincreased by incorporating a more rigid soft-segment, such aspolycarbonate diols.

The disclosed hydrophobic segmented block copolymer can also have anultimate tensile strength as measured by ASTM D1708 that is greater than6000 psi, e.g., greater than 8000 psi, greater than 8250 psi, greaterthan 8500 psi, or greater than 8750 psi. For example, the hydrophobicsegmented block copolymer can have an ultimate tensile strength from7900 to 8750 psi, from 8250 to 8750 psi, from 8500 to 8700 psi, or from7900 psi to 8250 psi.

In some embodiments, the base polymer can be synthesized to include apolyurethane membrane with both hydrophilic and hydrophobic regions tocontrol the diffusion of glucose and oxygen to an analyte sensor. Asuitable hydrophobic polymer component can be a polyurethane orpolyether urethane urea. Polyurethane is a polymer produced by thecondensation reaction of a diisocyanate and a difunctionalhydroxyl-containing material. A polyurea is a polymer produced by thecondensation reaction of a diisocyanate and a difunctionalamine-containing material. Preferred diisocyanates include aliphaticdiisocyanates containing from about 4 to about 8 methylene units.Diisocyanates containing cycloaliphatic moieties can also be useful inthe preparation of the polymer and copolymer components of the membranesof preferred embodiments. Examples of materials which can be used tomake non-polyurethane type membranes include vinyl polymers, polyethers,polyesters, polyamides, inorganic polymers such as polysiloxanes andpolycarbosiloxanes, natural polymers such as cellulosic and proteinbased materials, and copolymers, mixtures or combinations thereof.

In specific embodiments, the base polymer can be substantially free(e.g., less than 1 wt. %) of silicone.

In another embodiment of a suitable base polymer, a hydrophilic softsegment polymer component can be polyethylene oxide. For example, oneuseful hydrophilic copolymer component is a polyurethane polymer thatincludes about 20% hydrophilic polyethylene oxide. The polyethyleneoxide portions of the copolymer are thermodynamically driven to separatefrom the hydrophobic portions of the copolymer and the hydrophobicpolymer component. The 20% polyethylene oxide-based soft segment portionof the copolymer used to form the final blend affects the water pick-upand subsequent glucose permeability of the membrane. In this case, thelowest T_(g) of polymer domain formed of hydrophobic soft-segment orhard-segment is higher than −50° C.

Having a homogeneous segmented block copolymer can help ensure that thehydrophilic segments are more evenly distributed throughout thediffusion-resistance layer. The membrane's phase separation properties(or controlled location of hydrophilic/hydrophobic regions for creationof membrane permeation channels) can be controlled to a greater degreeby altering the size and length of the monomers/oligomers involved inthe membrane synthesis. Since, in this embodiment, only one polymer usedin the resistance layer, sensitivity change because of the hydrophilicpolymer leaching out will be minimum.

Alternatively, in some embodiments, the diffusion resistance layer cancomprise a combination or blend of a base polymer (e.g., segmentedpolyurethane block copolymers as disclosed herein) and one or morehydrophilic polymers (e.g., PVA, PEG, polyacrylamide, acetates, PEO,PEA, PVP, PDX, and, copolymers, blends, and/or variations thereof). Itis contemplated that any of a variety of combination of polymers can beused to yield a blend with desired glucose, oxygen, and interferencepermeability properties. The hydrophilic polymer can be blended with thebase polymer or can be covalently attached to the base polymer.

When the hydrophilic polymer is covalently attached to the base polymer,one way to create an aqueous dispersion is to dissolve the polymer inacetone and add the solution dropwise to an aqueous solvent mixing athigh shear. The aqueous solvent can be distilled water or water withstabilizing water-soluble additives such as PVP, salts, or SDS. Whileany variation can be used, one example for a diffusion-resistance layerwould use an aqueous dispersion with either an aqueous solvent of PVP inwater (0-50% PVP) with a PVP concentration that yields desired sensorsensitivity, or a pure water solvent to which a concentrated PVPsolution can be added after the dispersion is created.

In some specific embodiments, a tri-block polymer of PVP can be used asthe hydrophobic polymer in the diffusion-resistance layer. In stillother examples, the hydrophilic polymer can be a PVP that isfunctionalized with silane, alcohol, fluorine, or acrylate. In stillother examples, silica or similar nanocomposite materials can be used inthe blend in place of or in addition to the hydrophilic polymer. So, forexample, a diffusion-resistance layer can be formed from a blend of ahydrophobic block copolymer like a polycarbonate-urethane base polymeras disclosed herein and silica or a nanocomposite.

In yet further examples, the hydrophilic polymer can be crosslinkedwith, e.g., vinyl lactams. In a specific example, crosslinkedpolyvinylpyrrolidone (PVP) polymers can be made by reaction betweenepoxide-containing PVP copolymers and tertiary-amine-containing PVPcopolymers in solution at a predetermined temperature. Crosslinkablecopolymers of (a) 80-99% by wt. vinylpyrrolidone (VP) and 1-20% by wt.of a tertiary-amine-containing polymerizable monomer, e.g.vinylimidazole (VI) or 4-vinylpyridine (VPy), and (b) 80-99% by wt. VPand 1-20% by wt. of an epoxide-containing polymerizable monomer, e.g.allyl glycidyl ether (AGE) or glycidyl acrylate (GA), are reacted insolution, e.g. water, alcohol, or mixtures thereof, at a predeterminedtemperature, e.g. 50°-70° C., in a wt. ratio (solids basis) of (a):(b)of about 2:1 to 1:2, preferably about 1:1, in a solution concentrationof about 10-30% of each, to provide a crosslinked PVP product. Inanother example, a diffusion-resistance layer can be formed from a blendof a hydrophobic block copolymer like a polycarbonate-urethane basepolymer as disclosed herein and a crosslinked PVP and/or PVPfunctionalized with silanes, alcohol, or fluorine.

In some embodiments, the diffusion-resistance layer can be formed from ablend of a polycarbonate-urethane base polymer as disclosed herein and aPVP hydrophilic polymer. In some of the embodiments involving the use ofPVP, the PVP portion of the polymer blend can comprise from about 5% toabout 50% by weight of the polymer blend, sometimes from about 15% toabout 20%, and other times from about 25% to about 40%. It iscontemplated that PVP of various molecular weights can be used. Forexample, in some embodiments, the molecular weight of the PVP used canbe from about 25,000 daltons to about 5,000,000 daltons, sometimes fromabout 50,000 daltons to about 2,000,000 daltons, and other times fromabout 6,000,000 daltons to about 10,000,000 daltons. The samepercentages can apply when using PDX instead of PVP.

In some other embodiments, the base polymer can be endcapped with groupsthat can be used to link polymers together (so called networkingmoieties) and groups that can bloom to the surface and impact thesurface properties (so called blooming moieties). These are shown in theschematic of FIG. 49, where FG1 is a blooming moiety and FG2 is anetworking moiety.

It is also contemplated that the blooming moiety and networking moietycan be the same, as is shown in FIG. 50 where the blooming andnetworking moiety is a siloxane.

In other examples, the networking and blooming moieties are different,as is shown in FIG. 51, where the blooming moiety istris(trimethylsilyl)siloxane and the networking moiety ismethacrylamide, or in FIG. 52, where the blooming moiety istris(trimethylsilyl)siloxane and the networking moiety is carboxylicacid.

Additional examples of blooming moieties are fluorocarbons. Additionalexamples of networking moieties are vinyl groups (e.g., vinylalcohol,vinylbenzyl) isocyanates, amines, amides, alcohols, azides, thiols,alkenes, alkynes, esters, and the like. In some other embodiments, thenetworking moiety can be groups that complex iron (Fe(II) or Fe(III)).

Further, the disclosed diffusion-resistance layer can have zwitterionsentrapped or embedded within the polymer network by non-covalentinteractions. Thus, in further embodiments, the discloseddiffusion-resistance layer can comprise a base polymer and a hydrophilicpolymer and additional betaines blended therewith. For example, thediffusion-resistance layer can be blended with cocamidopropyl betaine,oleamidopropyl betaine, octyl sulfobetaine, caprylyl sulfobetaine,lauryl sulfobetaine, myristyl sulfobetaine, palmityl sulfobetaine,stearyl sulfobetaine, betaine (trimethylglycine), octyl betaine,phosphatidylcholine, glycine betaine, poly(carboxybetaine) (pCB), andpoly(sulfobetaine) (pSB). It will be appreciated that many morezwitterionic compounds or precursors or derivatives thereof can beapplicable and that this list of exemplary betaines is not intended tolimit the scope of the embodiments.

Sensors containing the disclosed diffusion-resistance layers can have aminimum drift (e.g., ≤10%) over extended time. For example, disclosedherein are sensors that have a drift of less than 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or 1% over 10 days.

A sensor having a diffusion resistance layer is shown as 46 in FIGS. 2Aand 2B; there it is situated more proximal to the implantable devicerelative to the biointerface layer. In some embodiments, thefunctionality of the diffusion resistance domain can be built into thebiointerface layer. Accordingly, it is to be noted that the descriptionherein of the diffusion resistance domain can also apply to thebiointerface layer. The diffusion resistance domain serves to controlthe flux of oxygen and other analytes (for example, glucose) to theunderlying enzyme domain.

The diffusion resistance domain 46 includes a semipermeable membranethat controls the flux of oxygen and glucose to the underlying enzymedomain 42, preferably rendering oxygen in non-rate-limiting excess. As aresult, the upper limit of linearity of glucose measurement is extendedto a much higher value than that which is achieved without the diffusionresistance domain. In some embodiments, the diffusion resistance domainexhibits an oxygen-to-glucose permeability ratio of approximately 200:1,but in other embodiments the oxygen-to-glucose permeability ratio can beapproximately 100:1, 125:1, 130:1, 135:1, 150:1, 175:1, 225:1, 250:1,275:1, 300:1, or 500:1. As a result of the high oxygen-to-glucosepermeability ratio, one-dimensional reactant diffusion can providesufficient excess oxygen at all reasonable glucose and oxygenconcentrations found in the subcutaneous matrix (see Rhodes et al.,Anal. Chem., 66:1520-1529 (1994)). In some embodiments, a lower ratio ofoxygen-to-glucose can be sufficient to provide excess oxygen by using ahigh oxygen soluble domain (for example, a silicone material) to enhancethe supply/transport of oxygen to the enzyme membrane or electroactivesurfaces. By enhancing the oxygen supply through the use of a siliconecomposition, for example, glucose concentration can be less of alimiting factor. In other words, if more oxygen is supplied to theenzyme or electroactive surfaces, then more glucose can also be suppliedto the enzyme without creating an oxygen rate-limiting excess.

In some embodiments, the diffusion-resistance layer 46 can be formed asa unitary structure with the biointerface domain 48; that is, theinherent properties of the diffusion resistance domain 46 areincorporated into biointerface domain 48 such that the biointerfacedomain 48 functions as a diffusion resistance domain 46.

In some embodiments, a diffusion resistance layer 46 can be used and canbe situated more proximal to the implantable device relative to thebiointerface layer. In some embodiments, the functionality of thediffusion resistance domain can be built into the biointerface layerthat comprises the polyzwitterionic biointerface polymer. Accordingly,it is to be noted that the description herein of the diffusionresistance domain can also apply to the biointerface layer. Thediffusion resistance domain serves to control the flux of oxygen andother analytes (for example, glucose) to the underlying enzyme domain.As described in more detail elsewhere herein, there exists a molarexcess of glucose relative to the amount of oxygen in blood, i.e., forevery free oxygen molecule in extracellular fluid, there are typicallymore than 100 glucose molecules present (see Updike et al., DiabetesCare 5:207-21 (1982)). However, an immobilized enzyme-based sensoremploying oxygen as cofactor is supplied with oxygen innon-rate-limiting excess in order to respond linearly to changes inglucose concentration, while not responding to changes in oxygentension. More specifically, when a glucose-monitoring reaction isoxygen-limited, linearity is not achieved above minimal concentrationsof glucose. Without a semipermeable membrane situated over the enzymedomain to control the flux of glucose and oxygen, a linear response toglucose levels can be obtained only up to about 40 mg/dL. However, in aclinical setting, a linear response to glucose levels is desirable up toat least about 500 mg/dL.

The diffusion resistance domain 46 includes a semipermeable membranethat controls the flux of oxygen and glucose to the underlying enzymedomain 44, preferably rendering oxygen in non-rate-limiting excess. As aresult, the upper limit of linearity of glucose measurement is extendedto a much higher value than that which is achieved without the diffusionresistance domain. In some embodiments, the diffusion resistance domainexhibits an oxygen-to-glucose permeability ratio of approximately 200:1,but in other embodiments the oxygen-to-glucose permeability ratio can beapproximately 100:1, 125:1, 130:1, 135:1, 150:1, 175:1, 225:1, 250:1,275:1, 300:1, or 500:1. As a result of the high oxygen-to-glucosepermeability ratio, one-dimensional reactant diffusion can providesufficient excess oxygen at all reasonable glucose and oxygenconcentrations found in the subcutaneous matrix (see Rhodes et al.,Anal. Chem., 66:1520-1529 (1994)). In some embodiments, a lower ratio ofoxygen-to-glucose can be sufficient to provide excess oxygen by using ahigh oxygen soluble domain (for example, a silicone material) to enhancethe supply/transport of oxygen to the enzyme membrane or electroactivesurfaces. By enhancing the oxygen supply through the use of a siliconecomposition, for example, glucose concentration can be less of alimiting factor. In other words, if more oxygen is supplied to theenzyme or electroactive surfaces, then more glucose can also be suppliedto the enzyme without creating an oxygen rate-limiting excess.

In some embodiments, the diffusion resistance domain is formed of a basepolymer synthesized to include a polyurethane membrane with bothhydrophilic and hydrophobic regions to control the diffusion of glucoseand oxygen to an analyte sensor. A suitable hydrophobic polymercomponent can be a polyurethane or polyether urethane urea. Polyurethaneis a polymer produced by the condensation reaction of a diisocyanate anda difunctional hydroxyl-containing material. A polyurea is a polymerproduced by the condensation reaction of a diisocyanate and adifunctional amine-containing material. Preferred diisocyanates includealiphatic diisocyanates containing from about 4 to about 8 methyleneunits. Diisocyanates containing cycloaliphatic moieties can also beuseful in the preparation of the polymer and copolymer components of themembranes of preferred embodiments. The material that forms the basis ofthe hydrophobic matrix of the diffusion resistance domain can be any ofthose known in the art as appropriate for use as membranes in sensordevices and as having sufficient permeability to allow relevantcompounds to pass through it, for example, to allow an oxygen moleculeto pass through the membrane from the sample under examination in orderto reach the active enzyme or electrochemical electrodes. Examples ofmaterials which can be used to make non-polyurethane type membranesinclude vinyl polymers, polyethers, polyesters, polyamides, inorganicpolymers such as polysiloxanes and polycarbosiloxanes, natural polymerssuch as cellulosic and protein based materials, and mixtures orcombinations thereof.

In one embodiment of a polyurethane-based resistance domain, ahydrophilic soft segment polymer component can be polyethylene oxide.For example, one useful hydrophilic copolymer component is apolyurethane polymer that includes about 20% hydrophilic polyethyleneoxide. The polyethylene oxide portions of the copolymer arethermodynamically driven to separate from the hydrophobic portions ofthe copolymer and the hydrophobic polymer component. The 20%polyethylene oxide-based soft segment portion of the copolymer used toform the final blend affects the water pick-up and subsequent glucosepermeability of the membrane.

Alternatively, in some embodiments, the diffusion resistance domain cancomprise a combination of a base polymer (e.g., polyurethane) and one ormore hydrophilic polymers (e.g., PVA, PEG, polyacrylamide, acetates,PEO, PEA, PVP, and variations thereof). It is contemplated that any of avariety of combination of polymers can be used to yield a blend withdesired glucose, oxygen, and interference permeability properties. Forexample, in some embodiments, the diffusion resistance domain can beformed from a blend of a silicone polycarbonate-urethane base polymerand a PVP hydrophilic polymer, but in other embodiments, a blend of apolyurethane, or another base polymer, and one or more hydrophilicpolymers can be used instead. In some of the embodiments involving theuse of PVP, the PVP portion of the polymer blend can comprise from about5% to about 50% by weight of the polymer blend, sometimes from about 15%to about 20%, and other times from about 25% to about 40%. It iscontemplated that PVP of various molecular weights can be used. Forexample, in some embodiments, the molecular weight of the PVP used canbe from about 25,000 daltons to about 5,000,000 daltons, sometimes fromabout 50,000 daltons to about 2,000,000 daltons, and other times fromabout 6,000,000 daltons to about 10,000,000 daltons.

In some embodiments, the diffusion resistance domain 46 can be formed asa unitary structure with the biointerface domain 48; that is, theinherent properties of the diffusion resistance domain 46 areincorporated into biointerface domain 48 such that the biointerfacedomain 48 functions as a diffusion resistance domain 46.

In certain embodiments, the thickness of the diffusion resistance domaincan be from about 0.05 μm or less to about 200 μm or more. In some ofthese embodiments, the thickness of the diffusion resistance domain canbe from about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 1, about 1.5,about 2, about 2.5, about 3, about 3.5, about 4, about 6, about 8 μm toabout 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 19.5, about 20, about 30,about 40, about 50, about 60, about 70, about 75, about 80, about 85,about 90, about 95, or about 100 μm. In some embodiments, the thicknessof the diffusion resistance domain is from about 2, about 2.5, or about3 μm to about 3.5, about 4, about 4.5, or about 5 μm in the case of atranscutaneously implanted sensor or from about 20 or about 25 μm toabout 40 or about 50 μm in the case of a wholly implanted sensor.

Enzyme Domain

The enzyme layer, also referred to as an enzyme domain, is the domain orlayer of an implantable device configured to immobilize an activeenzyme, which reacts with an analyte, when implanted in a host orconnected to the host (e.g., via an intravascular access deviceproviding extracorporeal access to a blood vessel). In some embodiments,the enzyme domain may be formed of an enzyme resistance domain describedin U.S. Provisional Application No. 62/273,155, filed Dec. 30, 2015,which is hereby incorporated by reference herein in its entirety.

The enzyme layers disclosed herein can be mechanically robust, resistphysiochemical degradation upon implantation, and withstand adhesivedegradation during the sensor implantation. Further, the disclosedenzyme layers do not affect the response time of the sensor, permeableto analytes, and do not alter the glucose rate limiting control byresistance layer. The disclosed enzyme layers can have hydrophilicproperties that have a water uptake of great than 10% relative to thedry weight, and fast water uptake and quick in reaching stabilization,so that sensor start-up is not affected negatively.

In some embodiments, the enzyme domain can comprise one surface-activeend group containing polymer, or a blend of two or more (e.g., two,three, four, five, or more) surface-active end group-containingpolymers, as described above. For example, in some embodiments theenzyme domain can comprise one surface-active end group containingpolymer that comprises surface-active end groups that are zwitterionic,or are precursors or derivatives thereof. In other embodiments, onesurface-active group containing polymer in a blend of two or moresurface-active group containing polymers comprises zwitterionicsurface-active groups, or precursors or derivatives thereof. In otherembodiments, a blend can comprise a polymer with positively chargedsurface-active groups and a polymer with negatively chargedsurface-active groups.

In some embodiments where the enzyme domain comprises one or morezwitterionic surface-active groups, or precursors or derivativesthereof, the zwitterionic surface-active group can comprise a betainemoiety such as a carboxyl, sulfo, or phosphor betaine group, orprecursors or derivatives thereof (for example alkylbetaines oraminobetaines), for example up to about 0.1, about 0.2, about 0.5, about1, about 2, or about 5% wt. of the domain. Exemplary betaines includecocamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine,caprylyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine,palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine),octyl betaine, phosphatidylcholine, glycine betaine,poly(carboxybetaine) (pCB), and poly(sulfobetaine) (pSB). It will beappreciated that many more zwitterionic groups, or precursors orderivatives thereof, can be applicable and that this list of exemplarybetaines is not intended to limit the scope of the embodiments. In someembodiments, hydrolyzable cationic esters of zwitterionic groups (asdiscussed elsewhere) can be used at similar concentrations forincorporation into the enzyme domain.

In some other embodiments, a blend of two or more surface-activegroup-containing polymers comprises one surface-active group that isnegatively charged and one surface-active group that is positivelycharged. In some embodiments, the number of negatively and positivelycharged surface-active groups is such that an enzyme domain formed fromthe blend is about net neutrally charged. In other embodiments, thenumber of positively charged and negatively charged surface-activegroups can be unequal, with either more positively charged or negativelycharged surface-active groups being present.

In some embodiments, the catalyst (enzyme) can be impregnated orotherwise immobilized into the biointerface or diffusion resistancedomain such that a separate enzyme domain 44 is not required (e.g.,wherein a unitary domain is provided including the functionality of thebiointerface domain, diffusion resistance domain, and enzyme domain). Insome embodiments, the enzyme domain 44 is formed from a polyurethane,for example, aqueous dispersions of colloidal polyurethane polymersincluding the enzyme.

In some embodiments, the enzyme layers disclosed herein may comprise anenzyme layer polymer. The enzyme layer polymer may be a polyzwitterion.Polyzwitterions are polymers where a repeating unit of the polymer chainis a zwitterionic moiety. As such, these polymers have the same numberof cationic and anionic groups, due to each zwitterion repeating unithaving both a positive and negative charge, and thus have an overallcharge of zero, often through a wide pH range. While not wishing to bebound by theory, it is believed that zwitterion groups in the enzymelayer polymer provide a charge center for strong charge-chargeinteraction with ionic groups in enzyme and can help immobilize theenzymes in the enzyme layer and decrease leaching of enzymes out of theenzyme layer (and sometimes into the host). Further, the zwitterionicgroups are highly hydrophilic and retain water and help prevent enzymefrom denaturing.

The enzyme layer polymers can be polyzwitterions, which aredistinguishable from other polyampholytes in that, which polyampholytescontain anionic and cationic groups, the ionic groups are not correlatedwith one another as part of the same repeating unit. So the anionic andcationic groups may be distributed apart from one another, at randomintervals, or one ionic group may outnumber the other. It is thustypical for a polyampholyte to have a net charge, except perhaps at somenarrow pH range.

The disclosed polyzwitterions can have a variety of repeating units,which are illustrated as i) through vii) below, where n is some integerfrom 2 to 1000:

In structures i) through iv) the zwitterionic unit is connected to thebackbone (

) and the charges are on side-groups that are pendant to the chain. Instructures v) through vii) the zwitterionic unit is such that one orboth charges is on the chain itself.

Examples of suitable zwitterionic monomers that can be used to produce apolyzwitterion of any of structures i) through vii) include:

-   -   ammoniophosphates (phosphobetaines or lecithin analogues),        ammoniophosphonates (phosphonobetaines), or ammoniophosphinates        (phosphinobetaines), respectively having the structures

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R², R³, and R⁴ are independently chosen from        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; wherein one or more of R¹, R², R³, R⁴, and Z are        substituted with a polymerization group.

By “polymerization group,” it is meant a functional group that permitspolymerization of the monomer with itself to form a homopolymer ortogether with different monomers to form a copolymer. Depending on thetype of polymerization methods employed, the polymerization group can beselected from alkene, alkyne, epoxide, lactone, amine, hydroxyl,isocyanate, carboxylic acid, anhydride, silane, halide, aldehyde, andcarbodiimide. In a step-growth polymerization, a matching pair offunctional groups are selected to promote polymerization, for example,to polymerize a zwitterionic monomer bearing di-hydroxyl group, adiisocyanate, epoxide, or di-carboxylic acid group containing co-monomercan be chosen to afford polymers formed with urethane, ether, and esterlinkages.

Further examples of suitable zwitterionic monomers that can be used toproduce a polyzwitterion of any of structures i) through vii) includeammoniosulfonates (sulfobetaines), ammoniosulfates, respectively havingthe structures:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³, are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group; and    -   ammoniocarboxylates having the structures:

-   -   where Z is branched or straight chain alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or        heteroaryl; and R² and R³ are independently chosen from alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;        wherein one or more of R¹, R², R³, and Z are substituted with a        polymerization group.

In each of these monomers Z can have a length of from 1 to 12 atoms,e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 atoms, where any ofthese values can form an upper or lower endpoint of a range.

These monomers can be prepared by methods known to those of skilled inthe art, e.g., as detailed in Laschewsky, “Structures and synthesis ofzwitterionic polymers,” Polymers 6:1544-1601, 2014. In certain examples,the disclosed polyzwitterions can have repeating zwitterionic unitsobtained from any of the zwitterionic monomers disclosed above.

The enzyme layer polymer may also comprises polyurethane and/or polyureasegments. For example, the enzyme layer polymer can comprise apolyurethane copolymer such as polyether-urethane-urea,polycarbonate-urethane, polyether-urethane, silicone-polyether-urethane,silicone-polycarbonate-urethane, polyester-urethane, polyurethane-urea,and the like. Since these polyurethane and/or polyurea segments containurea and/or urethane bonds formed from polyisocyanate and short chainpolyol or polyamine, which are hydrogen bonding rich moieties, thesesegments are referred to herein as “hard segments.” These segments canalso be relatively hydrophobic.

In addition to polyurethane and/or polyurea hard segments, the disclosedenzyme layer polymers can also comprise soft segments, which haverelatively poor hydrogen bonding. Soft segment are usually composed ofpolyols of polycarbonates, polyesters, polyethers, polyarylene, andpolyalkylene, and the like. The soft segments can be either hydrophobicor hydrophilic.

Enzyme layer polymers useful for certain embodiments can include linearor branched polymers on the backbone structure of the polymer. Thus,either the hard or soft segments can contain branching or linearbackbones.

The zwitterionic monomers can be part of either the hard or softsegments, or both, as described herein.

In some embodiments, the hard segment portion of the enzyme layerpolymer can comprise from about 5% to about 50% by weight of thepolymer, sometimes from about 15% to 20%, and other times from about 25%to 40%. The hard segments can have a molecular weight of from about 160daltons to about 10,000 daltons, and sometimes from about 200 daltons toabout 2,000 daltons. In some embodiments, the molecular weight of thesoft segments can be from about 200 daltons to about 10,000,000 daltons,and sometimes from about 500 daltons to about 5,000 daltons, andsometimes from about 500 daltons to about 2,000 daltons.

As noted the hard segments can be polyurethanes or polyureas.Polyurethane is a polymer produced by the condensation reaction of adiisocyanate and a difunctional hydroxyl-containing material. A polyureais a polymer produced by the condensation reaction of a diisocyanate anda difunctional amine-containing material. Preferred diisocyanatesinclude aliphatic diisocyanates containing from about 4 to about 9methylene units. Diisocyanates containing cycloaliphatic moieties canalso be useful in the preparation of the polymer and copolymercomponents of the membranes of preferred embodiments.

The soft segments used in the preparation of the enzyme layer polymercan be a polyfunctional aliphatic polyol, a polyfunctional aliphatic oraromatic amine, or the like that can be useful for creating permeabilityof the analyte (e.g., glucose) therethrough, and can include, forexample, polyoxazoline, poly(ethylene glycol) (PEG), polyacrylamide,polyimine,polypropylene oxide (PPO), PEG-co-PPO diol, silicone-co-PEGdiol, Silicone-co-PPO diol, polyethylacrylate (PEA),polyvinylpyrrolidone (PVP), and variations thereof (e.g., PVP vinylacetate), and wherein PEG and variations thereof can be preferred fortheir hydrophilicity.

In some of the embodiments, the soft segment portion of the enzyme layerpolymer can comprise from about 5% to about 70% by weight of thepolymer, sometimes from about 15% to 20%, and other times from about 25%to 40%. The soft segments can have a molecular weight of from about 160daltons to about 10,000 daltons, and sometimes from about 200 daltons toabout 2,000 daltons. In some embodiments, the molecular weight of thesoft segments can be from about 200 daltons to about 10,000,000 daltons,and sometimes from about 500 daltons to about 5,000 daltons, andsometimes from about 500 daltons to about 2,000 daltons.

In some embodiments, the enzyme layer polymer, including hard and softsegments, and zwitterionic repeating units, can have a molecular weightof from about 10 kDa to about 500,000 kDa, for example, from about 10kDa to about 100,000 kDa, from about 1000 kDa to about 500,000 kDa, fromabout 10,000 kDa to about 100,000 kDa, and from about 100,000 kDa toabout 500,000 kDa.

The hard and soft segments can each be selected for their properties,such as, but not limited to, tensile strength, flex life, modulus, andthe like. For example, polyurethanes are relatively strong and providenumerous reactive pathways, properties can be advantageous for amembrane domain of the continuous sensor.

As noted, the enzyme layer polymer contains one or more zwitterionicrepeating units; thus these groups are “internal” in reference to thepolymer backbone. Such “internal” repeating units are distinguished froma material that is found at the end of a polymer chain since such amoiety would only be bonded to the polymer chain at one location. Thedisclosed enzyme layer polymers can, in some embodiments, have one ormore zwitterionic groups at the terminal ends of the polymer chains;however, such groups are not the only zwitterionic groups in the chain;there is at least one internal zwitterionic group in the backbone.

In some embodiments, the enzyme layer polymer includes at least about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,about 28%, about 29%, about 30%, to about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54% or about 55% zwitterionic repeating units by weight ofthe polymer. In a preferred example, the enzyme layer polymer includesat least about 20% zwitterionic repeating units by weight of thepolymer.

The zwitterionic repeating unit can be a betaine such as a carboxyl,sulfo, or phosphor betaine compound, precursor or derivative thereof(for example alkylbetaines or aminobetaines). These segments or moietiescan be incorporated into the enzyme layer polymer, whether in the hardsegment, the soft segment, or both, for example up to about 55 wt. % ofthe enzyme layer polymer.

Although in some embodiments, using two or more different zwitterion orzwitterion precursor segments or moieties are used, in otherembodiments, a single zwitterion or zwitterion precursor segment ormoiety can be used in the enzyme layer polymer.

Some examples of an enzyme layer polymer are schematically illustratedin FIG. 4. Generally, the enzyme layer polymer comprises one or morehard segments and one or more soft segments. The hard segments can bealiphatic or aromatic monomers. The soft segments can be hydrophilic orhydrophobic oligomers of, for example, polyalkylene glycols,polycarbonates, polyesters, polyethers, polyvinylalcohol,polyvinypyrrolidone, polyoxazoline, and the like. The zwitterionicgroups (e.g., betaines) can be part of the soft segment, the hardsegments, or both. As illustrated, in FIG. 4, various hard and softsegments can be present, which permits one to tune the properties of theenzyme layer polymer by using different segments, different segmentslengths, functionalization on certain segments, crosslinking certainsegments, and the like. In some embodiments, biocompatible segmentedblock polyurethane copolymers comprising hard and soft segments can beused for the enzyme layer.

Incorporation of these zwitterionic repeating units into a polymer canbe achieved by using zwitterionic monomers that have diols or diamines(e.g., at position Z), or can be attached to diols or diamines at any ofR¹ through R⁴. Attaching a diol or diamine at R¹-R⁴ can be accomplishedby reacting the corresponding precursor with a halo-substituted diamineor halo-substituted diol. Examples of such monomers are shown below:

-   -   where W, Y, and Z are, independently, branched or straight chain        alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or        heteroaryl, any of which can be optionally substituted with O,        OH, halogen, amido, or alkoxyl; R¹ is H, alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; and R², R³,        and R⁴, are independently chosen from alkyl, heteroalkyl,        cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl. In specific        examples W is C₁-C₄ alkyl. In specific examples Y is C₁-C₄        alkyl. In other examples Z is C₁-C₄ alkyl.

These compounds can be reacted with a diisocyanate to form apolyurethane or polyurea. Alternatively, the carboxylates, sulfonates,phosphinates, or phophonates moieties can be protected and then theprotection group can be removed after polymerization. In anotheralternative, the amine can be a tertiary amine, which is thenquaternized by alkylation after polymerization.

Another method involves the radical polymerization of zwitterionicmonomers having unsaturated moieties substituted at position Z in themonomers shown above. In other examples zwitterionic monomers where anunsaturated moiety is attached to the ammonium group can be used in aradical polymerization. Examples of such monomers are shown below:

-   -   where X is O, NH, or NR⁴, Y and Z are, independently, branched        or straight chain alkyl, heteroalkyl, cycloalkyl,        cycloheteroalkyl, aryl, or heteroaryl, and of which can be        optionally substituted with OH, halogen, or alkoxyl; R¹ is H,        alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or        heteroaryl; and R³ and R⁵ are independently chosen from        heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl.        In specific examples, R⁵ is H or CH₃. In other examples, X is O.        In still other examples, X is NH or NCH₃. In specific examples Y        is C₁-C₄ alkyl. In other examples Z is C₁-C₄ alkyl.

Additional examples of suitable zwitterionic monomers includeN-(2-methacryloyloxy)ethyl-N,N-dimethylammonio propanesulfonate,N-(3-methacryloylimino)propyl-N,N-dimethylammonio propanesulfonate,2-(methacryloyloxy)ethylphosphatidylcholine, and3-(2′-vinyl-pyridinio)propanesulfonate.

In other embodiments, the enzyme layer polymer can be crosslinked. Forexample, polyurethaneurea polymers with aromatic or aliphatic segmentshaving electrophilic functional groups (e.g., carbonyl, aldehyde,anhydride, ester, amide, isocyanate, epoxy, allyl, or halo groups) canbe crosslinked with a crosslinking agent that has multiple nucleophilicgroups (e.g., hydroxyl, amine, urea, urethane, or thio groups). Infurther embodiments, polyurethaneurea polymers having aromatic oraliphatic segments having nucleophilic functional groups can becrosslinked with a crosslinking agent that has multiple electrophilicgroups. Still further, polyurethaneurea polymers having hydrophilicsegments having nucleophilic or electrophilic functional groups can becrosslinked with a crosslinking agent that has multiple electrophilic ornucleophilic groups. Unsaturated functional groups on the polyurethaneurea can also be used for crosslinking by reacting with multivalent freeradical agents.

Non-limiting examples of suitable cross-linking agents includeisocyanate, carbodiimide, gluteraldehyde or other aldehydes, aziridine,silane, epoxy, acrylates, free-radical based agents, ethylene glycoldiglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE),or dicumyl peroxide (DCP). In one embodiment, from about 0.1% to about15% w/w of cross-linking agent is added relative to the total dryweights of cross-linking agent and polymers added when blending theingredients (in one example, about 1% to about 10%). During the curingprocess, substantially all of the cross-linking agent is believed toreact, leaving substantially no detectable unreacted cross-linking agentin the final layer.

Further, the disclosed enzyme layer can have zwitterions entrapped orembedded within the polymer network by non-covalent interactions. Thus,in further embodiments, the disclosed enzyme layer can comprise anenzyme layer polymer and additional betaines blended therewith. Forexample, the enzyme layer polymer can be blended with cocamidopropylbetaine, oleamidopropyl betaine, octyl sulfobetaine, caprylylsulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmitylsulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octylbetaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine)(pCB), and poly(sulfobetaine) (pSB). It will be appreciated that manymore zwitterionic compounds or precursors or derivatives thereof can beapplicable and that this list of exemplary betaines is not intended tolimit the scope of the embodiments.

In certain embodiments, the enzyme layer can comprise thepolyzwitterionic enzyme layer polymer and the enzyme. In otherembodiments, the enzyme layer can comprise the polyzwitterionic enzymelayer polymer blended with a base polymer, and the enzyme. Suitable basepolymers may include, but are not limited to, silicone, epoxies,polyolefins, polystylene, polyoxymethylene, polysiloxanes, polyethers,polyacrylics, polymethacrylic, polyesters, polycarbonates, polyamide,poly(ether ketone), poly(ether imide), polyurethane, and polyurethaneurea, wherein polyurethanes and polyurethane urea may includepolyurethane copolymers such as polyether-urethane-urea,polycarbonate-urethane, polyether-urethane, silicone-polyether-urethane,silicone-polycarbonate-urethane, polyester-urethane, and the like. Insome embodiments, base polymers may be selected for their bulkproperties, such as, but not limited to, tensile strength, flex life,modulus, and the like. For example, polyurethanes are known to berelatively strong and to provide numerous reactive pathways, whichproperties may be advantageous as bulk properties for a membrane domainof the continuous sensor.

In some embodiments, a base polymer including biocompatible segmentedblock polyurethane copolymers comprising hard and soft segments may beused. In some embodiments, the hard segment of the copolymer may have amolecular weight of from about 160 daltons to about 10,000 daltons, andsometimes from about 200 daltons to about 2,000 daltons. In someembodiments, the molecular weight of the soft segment may be from about200 daltons to about 10,000,000 daltons, and sometimes from about 500daltons to about 5,000,000 daltons, and sometimes from about 500,00daltons to about 2,000,000 daltons. It is contemplated thatpolyisocyanates used for the preparation of the hard segments of thecopolymer may be aromatic or aliphatic diisocyanates. The soft segmentsused in the preparation of the polyurethane may be a polyfunctionalaliphatic polyol, a polyfunctional aliphatic or aromatic amine, or thelike that may be useful for creating permeability of the analyte (e.g.glucose) therethrough, and may include, for example, polyvinyl acetate(PVA), poly(ethylene glycol) (PEG), polyacrylamide, acetates,polyethylene oxide (PEO), polyethylacrylate (PEA), polyvinylpyrrolidone(PVP), Poly(2-oxazoline (PDX), and variations thereof (e.g. PVP vinylacetate), and wherein PVP, PDX and variations thereof may be preferredfor their hydrolytic stability in some embodiments.

In some embodiments, the one or more zwitterionic compounds orprecursors thereof applied to the surface of the membrane system arehydrolyzable cationic esters of zwitterionic compounds. In theseembodiments, the hydrolyzable cationic esters provide the added benefitthat hydrolysis of the cationic esters into nonfouling zwitterionicgroups can kill microbes (such as bacteria) or condense DNA. Further,the mixed-charge nature of the resulting zwitterionic groups result ininhibition of nonspecific protein adsorption on the surface of thesensors. In these embodiments, cationic betaine esters, such as cationicpCB esters are preferable.

It has been found that incorporation of zwitterion or zwitterionprecursor segments or moieties internally in the polymer backbone can bechallenging due to the solubility issues associated with the monomers ofthe zwitterion or zwitterion precursors. Such groups typically can onlybe dissolved in highly polar solvents such as methanol and water, whichare not favorable in the synthesis of some the enzyme layer polymers(e.g., polyurethanes). Thus, the available functional groups that couldbe used chemically incorporated into the enzyme layer polymer's backboneby solution based polycondensation synthesis was limited. As analternative method of incorporating zwitterion or zwitterion precursorsegments or moieties into the base-polymer's backbone, precursors orderivatives of the zwitterion or zwitterion precursors can be used. Forexample, zwitterion precursors and/or zwitterionic derivatives, whichhave more desirable solubility characteristics in low polarity organicsolvents, can be used as monomers. The enzyme layer polymer (e.g.,polyurethaneureas) can be synthesized by polycondensation reactions andform well-defined polymers with high molecular weight and lowpolydispersity index. These polymers can then be converted to zwitteriongroup containing polymers via chemical reaction (such as hydrolysis,deprotection, heat-triggered rearrangement, and UV-triggereddegradation) or biological triggered reaction after in vivo implantationof the device.

In some embodiments, enzymes included in an enzyme layer are susceptibleto thermal or pH induced degradation. In some related embodiments, theenzyme layer may also comprise one or more enzyme stabilizing agents.Such agents improve the enzyme's ability to resist thermal or pH induceddenaturing. Inclusion of enzyme stabilizing agents thus facilitatesdevice fabrication by allowing for use of fabrication processes whichwould otherwise compromise the enzyme's activity. Inclusion of theseagents has the added benefits of extending useable and shelf lives ofthe sensors. Any material which improves thermal and/or pH stability ofthe enzyme, without affecting analyte or oxygen permeability of theenzyme layer to the point that the enzyme layer is no longer suitablefor use in a sensor, may be used as an enzyme stabilizing agent. In someembodiments, an enzyme stabilizing agent may be dipolar. Without wishingto be bound by theory, it is believed that dipolar enzyme stabilizingagents stabilize the enzyme by orienting around the enzyme in such a wayas to provide a charged local environment that stabilizes the enzyme'stertiary structure.

Dipolar enzyme stabilizing agents may be zwitterionic ornon-zwitterionic. That is, dipolar enzyme stabilizing agents are neutralmolecules with a positive and negative electrical charge at differentlocations. In some embodiments, the positive and negative electricalcharges are full unit charges (i.e., the molecules are zwitterionic). Inother embodiments, the positive and negative charges are less than fullunit charges (i.e., the molecules are dipolar, but non-zwitterionic).

In some embodiments, a zwitterionic enzyme stabilizing agent may be abetaine, such as glycine betaine, poly(carboxybetaine) (pCB), orpoly(sulfobetaine) (pSB), or some other zwitterion, such ascocamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine,caprylyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine,palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine),octyl betaine, phosphatidylcholine, ectoine, or hydroxyectoine. Inpreferred embodiments, the zwitterionic enzyme stabilizing agent isglycine betaine. In some embodiments, a non-zwitterionic enzymestabilizing reagent may be an amine oxide.

The enzyme stabilizing reagents, if used, can be present at up to about0.1, about 0.2, about 0.5, about 1, about 2, or about 5% wt. of theenzyme layer. It will be appreciated that many more zwitterionic groups,or precursors or derivatives thereof, can be applicable and that thislist of exemplary betaines is not intended to limit the scope of theembodiments. In some embodiments, hydrolyzable cationic esters ofzwitterionic groups (as discussed elsewhere) can be used at similarconcentrations for incorporation into the enzyme layer.

In embodiments where the enzyme layer comprises an enzyme stabilizingreagent, the amount of enzyme stabilizing reagent present in the enzymedomain is sufficient to provide an improvement in the thermal and/or pHstability of the enzyme, while not disrupting the permeabilitycharacteristics of the enzyme layer so that the sensor retains highglucose sensitivity. The identity and amount of enzyme stabilizingreagent used in the enzyme layer may vary based on the particular enzymeused in the sensor; however, the amount of enzyme stabilizing reagent isgenerally less than about 50% wt. of the amount of the enzyme; such asless than about 25% wt; such as less than about 10 wt. %. In a preferredembodiment, the enzyme is glucose oxidase and the enzyme stabilizingreagent is a betaine, such as glycine betaine.

In some embodiments, the enzyme and enzyme layer polymer, and optionalenzyme stabilizing agents, can be impregnated or otherwise immobilizedinto the biointerface layer or diffusion resistance domain such that aseparate enzyme layer is not required (e.g. wherein a unitary domain isprovided including the functionality of the biointerface layer,diffusion resistance domain, interference domain, and enzyme layer). Insome embodiments, the enzyme layer is formed from a polyurethane, forexample, aqueous dispersions of colloidal polyurethane polymersincluding the enzyme and enzyme stabilizing reagent. Again, it iscontemplated that in some embodiments, the polymer system of the enzymelayer may not be crosslinked, but in other embodiments, crosslinking maybe used and achieved by any of a variety of methods, for example, byadding a crosslinking agent.

In some other embodiments, a blend of two or more surface-activegroup-containing polymers comprises one surface-active group that isnegatively charged and one surface-active group that is positivelycharged. In some embodiments, the number of negatively and positivelycharged surface-active groups is such that an enzyme domain formed fromthe blend is about net neutrally charged. In other embodiments, thenumber of positively charged and negatively charged surface-activegroups can be unequal, with either more positively charged or negativelycharged surface-active groups being present.

In some embodiments, disclosed are membranes that comprise an enzymelayer 44 (see FIGS. 2A through 2C). The enzyme layers disclosed hereincan comprise the enzyme layer polymer and an enzyme, or in analternative embodiment, the enzyme layer can comprise the enzyme layerpolymer and one or more other polymers, forming a polymer blend, and anenzyme.

In some embodiments, the enzyme layer 44, can be used and is situatedless distal from the electrochemically reactive surfaces than thediffusion resistance domain 46. The enzyme layer comprises an enzymeconfigured to react with an analyte. In one embodiment, the membranecomprises an immobilized enzyme layer 44 including glucose oxidase. Inother embodiments, the enzyme layer 44 can be impregnated with otheroxidases, for example, galactose oxidase, cholesterol oxidase, aminoacid oxidase, alcohol oxidase, lactate oxidase, or uricase. For example,for an enzyme-based electrochemical glucose sensor to perform well, thesensor's response should neither be limited by enzyme activity norcofactor concentration. In other embodiments, the enzyme may be adehydrogenase, such as a glucose dehydrogenase.

In some embodiments, the enzyme can be impregnated or otherwiseimmobilized into the biointerface or diffusion resistance domain suchthat a separate enzyme domain 44 is not required (e.g., wherein aunitary domain is provided including the functionality of thebiointerface domain, diffusion resistance domain, and enzyme domain). Insome embodiments, the enzyme domain 44 is formed from a polyurethane,for example, aqueous dispersions of colloidal polyurethane polymersincluding the enzyme.

In some embodiments, the thickness of the enzyme domain can be fromabout 0.01, about 0.05, about 0.6, about 0.7, or about 0.8 μm to about1, about 1.2, about 1.4, about 1.5, about 1.6, about 1.8, about 2, about2.1, about 2.2, about 2.5, about 3, about 4, about 5, about 6, about 8,about 10, about 15, about 20, about 30, about 40, about 50, about 60,about 70, about 75, about 80, about 90, about 100 μm, about 125, about150, about 175, about 200 or about 250 μm. In some of these embodiments,the thickness of the enzyme domain can be from about 0.05, about 0.1,about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4,about 0.45, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3,about 4, or about 5 μm to about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 19.5, about 20, about 25, or about 30 μm. Insome of these embodiments, the thickness of the enzyme layer can besometimes from about 1 to about 5 μm, and sometimes from about 2 toabout 7 μm. In other embodiments, the enzyme layer can be from about 20or about 25 μm to about 50, about 55, or about 60 μm thick. In evenfurther embodiments, the thickness of the enzyme domain is from about 2,about 2.5, or about 3 μm to about 3.5, about 4, about 4.5, or about 5μm; in the case of a transcutaneously implanted sensor is from about 6,about 7, or about 8 μm to about 9, about 10, about 11, or about 12 μm inthe case of a wholly implanted sensor. In some embodiments, the glucosesensor can be configured for transcutaneous or short-term subcutaneousimplantation, and can have a thickness from about 0.5 μm to about 8 μm,and sometimes from about 4 μm to about 6 μm. In one glucose sensorconfigured for fluid communication with a host's circulatory system, thethickness can be from about 1.5 μm to about 25 μm, and sometimes fromabout 3 to about 15 μm. It is also contemplated that in someembodiments, the enzyme layer or any other layer of the electrode canhave a thickness that is consistent, but in other embodiments, thethickness can vary. For example, in some embodiments, the thickness ofthe enzyme layer can vary along the longitudinal axis of the electrodeend.

In another aspect, the enzyme layer can have a biomimetic adhesivepolymer as an additive blended into the enzyme layer to enhance theadherence of the enzyme layer to the diffusion resistance domain andinterference domain and decrease delamination. Suitable biomimeticadhesive polymers that can be used in this embodiment are3,4-dihydroxy-L-phenylalanine containing polymers.3,4-dihydroxy-L-phenylalanine (DOPA), the active ingredient in marinemussel proteins, can be converted into polymerizable monomers and thempolymerized to form linear nondegradable homo or copolymers.

Interference Domain

It is contemplated that in some embodiments, such as in the sensorconfiguration illustrated in FIG. 2B, an interference domain 43, alsoreferred to as the interference layer, may be provided in addition to(or in replacement of) the biointerface layer. The interference domain43 may substantially reduce the permeation of one or more interferentsinto the electrochemically reactive surfaces. The interference domain 43can be configured to be much less permeable to one or more of theinterferents than to the measured species. It is also contemplated thatin some embodiments, where interferent blocking may be provided by thebiointerface layer (e.g., via a surface-active group-containing polymerof the biointerface layer), a separate interference domain is notpresent. In other embodiments, the membrane includes both aninterference domain and a biointerface layer, with both domainsconfigured to reduce the permeation of one or more interferents. Infurther embodiments, the interference domain and the biointerface layerare each configured to reduce permeation of different interferingspecies. For example, the interference domain may have greaterspecificity than the biointerface layer with respect to reducingpermeation of one type of interfering species, while the biointerfacelayer may have greater specificity than the interference domain withrespect to reducing permeation of another type of interfering species.In some embodiments, both the interference domain and the biointerfacelayer are configured to target certain interference species forpermeation reduction.

In certain embodiments, the implantable sensor employs a membrane systemcomprising a resistance domain, an enzyme domain, and an interferencedomain. The interference domain can be proximal to the sensor and theresistance domain can be distal to the sensor, with the enzyme domaintherebetween. The interference domain can consist of a single layer orplurality of layers of the same material. However, in some embodiments,the interference domain comprises two or more different types of layersin an alternating configuration. For example, a first type of layer canbe represented by X, a second type of layer can be represented by Y, anda third type of layer can be represented by Z. The interference domainincluding alternating layers can have the following exemplaryconfigurations:

XY YX XYX XYXYX XYXYXY XXYYXYXXYY XXXYYYXXXYYYXXX XYXYXYXYXYXYXXYZXYZXYZX XYXZXYXZXYXZ ZYYXZZZXYYYXZ

The above configurations, which are merely exemplary, illustrate variousembodiments. In certain embodiments, the first and last layers are thesame (e.g., X and X), in other embodiments, the first and last layersare different (e.g., X and Y). The domain can include one or more layersthat are unitary (i.e., a single layer is deposited, e.g., X), orcomposite (e.g., a first layer of material is deposited, followed by thedeposition of a second and third, etc. layer of the same material atopthe first layer, e.g., XXX). The pattern of alternating layers can beregular (e.g., XYXYXYXYXY) or irregular (e.g., ZYXZXYZYZ).

In some embodiments, the alternating layers include polyanionic layersand polycationic layers. The following are exemplary interference domainconfigurations, wherein the polyanionic layers (unitary, composite,and/or contiguous with the same polyanion or with different polyanions)are represented by A and the polycationic layers by C (unitary,composite, and/or contiguous with the same polyanion or with differentpolyanions):

CA CAC CACA CACAC CACACA CACACAC CACACACA CACACACAC CACACACACACACACACACAC CACACACACACA CACACACACACAC CACACACACACACA CACACACACACACACCACACACACACACACA CACACACACACACACAC CACACACACACACACACACACACACACACACACACAC CACACACACACACACACACA CACACACACACACACACACACCACACACACACACACACACACA CACACACACACACACACACACAC CACACACACACACACACACACACACACACACACACACACACACACACAC CACACACACACACACACACACACACACCACACACACACACACACACACACACACAC CACACACACACACACACACACACACACACACCACACACACACACACACACACACACACACACAC AC ACA ACAC ACACA ACACAC ACACACAACACACAC ACACACACA ACACACACAC ACACACACACA ACACACACACAC ACACACACACACAACACACACACACAC ACACACACACACACA ACACACACACACACAC ACACACACACACACACAACACACACACACACACAC ACACACACACACACACACA ACACACACACACACACACACACACACACACACACACACACA ACACACACACACACACACACAC ACACACACACACACACACACACAACACACACACACACACACACACAC ACACACACACACACACACACACACAACACACACACACACACACACACACACA ACACACACACACACACACACACACACACAACACACACACACACACACACACACACACACA ACACACACACACACACACACACACACACACACA

Other configurations (e.g., those including additional layers, and/oradditional materials) are also contemplated for some embodiments. Insome embodiments, each A layer is a unitary or composite layer of thesame polyanion, and each C layer is a unitary or composite layer of thesame polycation. The outermost layers of the interference domain canboth be polycation layers, with polyanion layers present only asinterior layers. Any suitable number of alternating layers can beemployed in the interference domain, for example, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bilayers(defined as a polycationic layer adjacent to a polyanionic layer). Insome embodiments a final polycationic layer is added so as to yield aninterference domain with polycationic layers as the outermost layers. Inother embodiments a final anionic layer is added so as to yield aninterference domain with polyanionic layers as the outermost layers.

Polyanions and polycations belong to the class of polymers commonlyreferred to as polyelectrolytes—polymers wherein at least some of therepeating units (or monomers) include one or more ionic moieties.Polyelectrolytes which bear both cationic and anionic moieties arecommonly referred to as polyampholytes. Certain polyelectrolytes formself-assembled monolayers wherein one end of the molecule shows aspecific, reversible affinity for a substrate such that an organized,close-packed monolayer of the polyelectrolyte can be deposited.

The polycation can be any biocompatible polycationic polymer. In someembodiments, the polycation is a biocompatible water-solublepolycationic polymer. In certain embodiments, water solubility may beenhanced by grafting the polycationic polymer with water-solublepolynonionic materials such as polyethylene glycol. Representativepolycationic materials may include, for example, natural and unnaturalpolyamino acids having a net positive charge at neutral pH, positivelycharged polysaccharides, and positively charged synthetic polymers.Additional examples of suitable polycationic materials includepolyamines having amine groups on either the polymer backbone or thepolymer sidechains, such as poly-L-lysine and other positively chargedpolyamino acids of natural or synthetic amino acids or mixtures of aminoacids, including poly(D-lysine), poly(ornithine), poly(arginine), andpoly(histidine), and nonpeptide polyamines such as poly(aminostyrene),poly(aminoacrylate), poly(N-methyl aminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),poly(N,N-diethylaminoacrylate), poly(diallyldimethyl ammonium chloride),poly(aminomethacrylate), poly(N-methyl amino-methacrylate), poly(N-ethylaminomethacrylate), poly(N,N-dimethyl aminomethacrylate),poly(N,N-diethyl aminomethacrylate), poly(ethyleneimine), polymers ofquaternary amines, such as poly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride), and natural orsynthetic polysaccharides such as chitosan, poly(allylaminehydrochloride), poly(diallyldimethylammonium chloride),poly(vinylbenzyltriamethylamine), polyaniline or sulfonated polyaniline,(p-type doped), polypyrrole (p-type doped), polyallylaminegluconolactone, and poly(pyridinium acetylene).

The polyanionic material can be any biocompatible polyanionic polymer,for example, any polymer having carboxylic acid groups attached aspendant groups. The polyionic layers can be hydrophilic (e.g., amaterial or portion thereof which will more readily associate with waterthan with lipids). In some embodiments, the polyanionic polymer is abiocompatible water-soluble polyanionic polymer. Suitable materialsinclude, but are not limited to, alginate, carrageenan, furcellaran,pectin, xanthan, hyaluronic acid, heparin, heparan sulfate, chondroitinsulfate, dermatan sulfate, dextran sulfate, polymethacrylic acid,polyacrylic acid, poly(vinyl sulfate), poly(thiophene-3-acetic acid),poly(-styrenesulfonic acid), poly(styrene sulfonate),(poly[1-[4-(3-carboxy-4-hydroxy-phenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt]),poly(4-[4-({4-[3-amino-2-(4-hydroxy-phenyl)propylcarbamoyl]-5-oxo-pentyl-}-methyl-amino)-phenylazo]-benzenesulfonicacid), oxidized cellulose, carboxymethyl cellulose and crosmarmelose,synthetic polymers, and copolymers containing pendant carboxyl groups,such as those containing maleic acid or fumaric acid in the backbone.Polyaminoacids of predominantly negative charge are also suitable.Examples of these materials include polyaspartic acid, polyglutamicacid, and copolymers thereof with other natural and unnatural aminoacids. Polyphenolic materials, such as tannins and lignins, can be usedif they are sufficiently biocompatible.

The molecular weight of the polyionic materials may be varied in orderto alter coating characteristics, such as coating thickness. As themolecular weight is increased, the coating thickness generallyincreases. However, an increase in molecular weight may result ingreater difficulty with handling. To achieve a balance of coatingthickness, material handling, and other design considerations, thepolyionic materials can have a particular average molecular weight Mn.In some embodiments, the average molecular weight of a polyionicmaterial used is from about 1,000, 10,000, or 20,000 to about 25,000,50,000, 100,000 or 150,000 g/mol.

In some embodiments, the interference domain can be prepared using alayer-by-layer deposition technique, wherein a substrate (e.g., thesensor or membrane layer atop the sensor, e.g., the resistance or enzymelayer) is dipped first in a bath of one polyelectrolyte, then in a bathof an oppositely charged polyelectrolyte. Optionally, the substrate canbe dipped in a bath of rinsing solution before or after the substrate isdipped into the polyelectrolyte bath. During each dip a small amount ofpolyelectrolyte is adsorbed and the surface charge is reversed, therebyallowing a gradual and controlled build-up of electrostaticallycross-linked films (or hydrogen bonded films) of alternatingpolycation-polyanion layers. The method provides a technique forcontrolling functionality and film thickness and functionality. Forexample, it can be employed for depositing films as thin as onemonolayer or for thicker layers. FIG. 26B illustrates one embodiment ofa layer-by-layer deposition method, which employs alternating adsorptionof polycations and polyanions to create a structure illustrated in FIG.26A. Operationally, the embodiment illustrated in FIG. 26B, occursthrough consecutive exposures of a substrate 938 to polycation andpolyanion solutions, with rinsing to remove unadsorbed polymer aftereach deposition step. In a first step, a polycation 942 is depositedonto a substrate 938 (e.g., a wire with an electroactive surface or aflat wafer substrate) to form a polycationic layer 942. As describedelsewhere herein in greater detail, the deposition of the layer can beperformed using any of a variety of techniques, such as, dipping and/orspraying, for example. In a second step, rinsing is performed to removeunadsorbed polymer after deposition of the polycationic layer 942. Next,in a third step, a polyanion 944 is deposited onto the polycationiclayer 942. Thereafter, in a fourth step, rinsing is performed to removeunadsorbed polymer after deposition of the polyanionic layer 944. Thesesteps can be repeated until the desired interference domainconfiguration and/or structure is achieved. In an alternativeembodiment, instead of depositing a polycationic layer as the firstlayer on top of the substrate 938, a polyanionic layer is depositedinstead. Thereafter, a second layer formed of a polycation is depositedonto the first layer, i.e., the polyanionic layer. This process iscontinued until a certain desired interference domain configurationand/or structure is achieved.

In some embodiments, methods can also employ other interactions such ashydrogen bonding or covalent linkages. Depending upon the nature of thepolyelectrolyte, polyelectrolyte bridging may occur, in which a singlepolyelectrolyte chain adsorbs to two (or more) oppositely chargedmacroions, thereby establishing molecular bridges. If only a monolayerof each polyelectrolyte adsorbs with each deposition step, thenelectrostatically cross-linked hydrogel-type materials can be built on asurface a few microns at a time. If the substrate is not thoroughlyrinsed between the application of polyionic films, thicker,hydrogel-like structures can be deposited.

In some embodiments, the interference blocking ability provided by thealternating polycationic layer(s) and polyanionic layer(s) can beadjusted and/or controlled by creating covalent cross-links between thepolycationic layer(s) and polyanionic layer(s). Cross-linking can have asubstantial effect on mechanical properties and structure of the film,which in turn can affect the film's interference blocking ability.Cross-linked polymers can have different cross-linking densities. Incertain embodiments, cross-linkers are used to promote cross-linkingbetween layers. In other embodiments, in replacement of (or in additionto) the cross-linking techniques described above, heat is used to formcross-linking. For example, in some embodiments, imide and amide bondscan be formed between a polycationic layer and a polyanionic layer as aresult of high temperature. In some embodiments, photo cross-linking isperformed to form covalent bonds between the polycationic layers(s) andpolyanionic layer(s). One major advantage to photo-cross-linking is thatit offers the possibility of patterning. In certain embodiments,patterning using photo-cross linking is performed to modify the filmstructure and thus to adjust the interference domain's interferenceblocking ability. Blocking ability can correspond to, but is not limitedto, the ability to reduce transport of a certain interfering species orto the selectivity for the transport of a desired species (e.g., H₂O₂)over an interfering species. Post-deposition reactions, such ascross-linking and reduction of metal ions to form nanoparticles, providefurther ways to modify film properties. In some embodiments,cross-linking may be performed between deposition of adjacentpolycationic or polyanionic layers in replacement of (or in addition to)a post-deposition cross-linking process.

The overall thickness of the interference layer can impact itspermeability to interferents. The overall thickness of the interferencedomain can be controlled by adjusting the number of layers and/or thedegree of rinsing between layers. With layer deposition throughspraying, control of drop size and density can provide coatings ofdesired selected thickness without necessarily requiring rinsing betweenlayers. Additionally, the excess (unbound) material can be removed viaother means, for example, by an air jet. If the residual polyelectrolytefrom the previous layer is substantially removed before adding thesubsequent layer, the thickness per layer decreases. Accordingly, in oneembodiment, the surface is first coated with a polycation, the excesspolycation is then removed by rinsing the surface, afterwards thepolyanion is added, the excess is then removed, and the process isrepeated as necessary. In some embodiments, the polycations orpolyanions from different adjacent layers may intertwine. In furtherembodiment, they may be intertwined over several layers.

In some embodiments, the level of ionization of polyions may becontrolled, for example, by controlling the pH in the dip solutioncomprising the polycation or the polyanion. By changing the level ofionization of these polyions, the interference blocking ability of acertain layer of may be altered and/or controlled. For example, a firstpolycationic layer that has a higher level of ionization than a secondpolycationic layer may be better at interacting with and reducing thetransport a first interfering species, while the second polycationic maybe better at interacting with and reducing the transport of a secondinterfering species. Changes in the level of ionization of a polyion'scharge groups can also affect the mechanical properties, structuralproperties, and other certain properties (e.g., diffusion properties)that may affect the interference domain's ability to reduce transport of(or entirely block) interfering species. For example, an alternatingbilayer, comprising polycations and polyanions, both of which have highlevels of ionization, may bond together more tightly than acorresponding bilayer with low levels of ionization. Thus, thestructural difference between these two membranes, which can be in theform of mechanical properties or other properties (e.g., thickness ofthe domain), can affect the performance of the interference domain.

In some embodiments, the linear charge density of the polyelectrolytemay be controlled at least in part by the average charge spacing alongthe polyion chain. The spacing between charge groups on the polycationicand/or polyanionic polymers that form the interference domain may becontrolled by polyelectrolyte polymer selection or polymer synthesis.How far the charged groups are spaced can greatly affect the structuralproperties of the interference domain. For example, a polyion havingcharged groups that are spaced closely to each other may result insmall-sized pores in the interference domain, thereby resulting in astructure that excludes medium molecular-sized and large molecular-sizedinterfering species from passage therethough, while allowing passagetherethrough of small-sized pores. Conversely, a polyion having chargedgroups that are spaced apart at a moderate distance from each other mayresult in medium-sized pores that exclude large molecular-sizedinterfering species and allow passage therethrough of medium-molecularsized and small molecular-sized interfering species. In certainembodiments, the linear charge density of the polyanionic polymer isfrom about 1 to 50 e/Å, sometimes from about 10 to 25 e/Å, sometimesfrom about 2 to 10 e/Å, and sometimes from 2 to 3 e/Å, where e is theelementary charge of an electron/proton and A is distance in angstroms.In some embodiments, the linear charge density of the polycationicpolymer is from about 1 to 50 e/Å, sometimes from about 10 to 15 e/Å,sometimes from about 2 to 10 e/Å, and sometimes from 2 to 3 e/Å.

In some embodiments, the linear charge density of polyanionic polymer issubstantially similar to the linear charge density of the polycationicpolymer. For example, in one embodiment, the polyanionic layer is formedof (i) poly(acrylic acid), which has an average linear charge density ofabout 2.5 e/Å and (ii) poly(allylamine hydrochloride), which also has anaverage linear charge density of about 2.5 e/Å. In certain embodiments,the polycationic and polyanionic layers may have an average linearcharge density that is substantially equal with each other and that isfrom about 1 to 50 e/Å, sometimes from about 2 to 25 e/Å, sometimes fromabout 5 to 10 e/Å, other times from about 10 to 15 e/Å, and other timesfrom about 15 to 25 e/Å.

By providing an interference domain with differing linear chargedensities, an interference domain may be formed that comprises differentpolycationic/polyanionic bilayers that are designed specifically toexclude different interfering species based on certain characteristics(e.g., molecular size) of the targeted interfering species. For example,in one embodiment, an outermost bilayer of the interference domain isdesigned to have a medium average charge spacing, thereby resulting in abilayer that only excludes large molecular-sized species, but allowspassage therethrough of medium molecular-sized species and smallmolecular-sized species. Conversely, an innermost bilayer of theinterference domain may be designed to have low average charge spacing,thereby resulting in a bilayer that excludes all molecules, except thosewith very small molecular sizes, for example, H₂O₂.

In some embodiments, the polycationic layers may be formed of the sameor substantially the same material (e.g., poly(allylamine hydrochloride)(PAH) for polycation or poly(acrylic acid) (PAA) for polyanion), whilehaving different levels of ionization. For example, in one embodiment,the interference domain comprises seven alternating polyelectrolytelayers, with the first, third, fifth, and seventh layers beingpolycationic layers, and with the second, fourth, and sixth layers beingpolyanionic layers, wherein the first and seventh layers form the outerlayers of the interference domain. In one embodiment, each or some ofthe polycationic layers may have different levels of ionization. Forexample, in one embodiment, the first, third, fifth, and seventh layersmay each have different levels of ionization, with the first layerhaving the highest level of ionization and the seventh layer having thelowest level of ionization, or vice versa. In an alternative embodiment,some of the polycationic layers may share substantially the same levelof ionization. For example, in one embodiment, the first and seventhlayers may have substantially the same levels of ionization, while thethird and fifth layers may have a level of ionization that is differentfrom the others. As described elsewhere herein, the ionization level ofa polyion may be controlled by controlling the pH in the dip solutioncomprising the polycation or the polyanion. By changing the level ofionization of these polyions, the interference blocking ability of acertain layer of may be altered and/or controlled.

The design of an interference domain having layers with levels ofionization can also be applied to polyanionic layers as well. Forexample, in one embodiment with seven alternating polyelectrolytelayers, the second, fourth, and sixth layers are each polyanionic layersand may each have different levels of ionization, with the second layerhaving the highest level of ionization and the sixth layer having thelowest level of ionization, or vice versa. In an alternative embodiment,some of the polyanionic layers may share substantially the same level ofionization. For example, in one embodiment, the second and fourth layersmay have substantially the same levels of ionization, while the sixthlayer may have a substantially different level of ionization from theothers.

In certain embodiments, the particular polycationic layer(s) and/orpolyanionic layer(s) selected to form the interference layer may dependat least in part on their ability to block, reduce, or impede passagetherethrough of one or more interferents. For example, the polyanioniclayer can be selected for its ability to block, reduce, or impedepassage of a first interferent, whereas the polycationic layer isselected for its ability to block, reduce, or impede passage of a secondinterferent. The layer may be designed to slow but not block passage ofan interferent therethrough, or designed to substantially block (e.g.,trap) an interferent therein. Additional polyionic layers can beincluded in the interference domain with particular selectivity towardsstill different interferents. Depending upon the position of theinterference domain in the membrane system relative to the electrode orelectroactive surface of the sensor, the permeability of the layer tosubstances other than the interferent can be important. In sensorsystems wherein H₂O₂ (hydrogen peroxide) is produced by anenzyme-catalyzed reaction of an analyte being detected, the interferencedomain should be designed to allow H₂O₂ to pass through with minimalimpedance if the interference domain is positioned between theelectroactive surface and the enzyme layer. On the other hand, if in adifferent membrane design, the interference domain is positioned distalto the enzyme layer (with respect to the electroactive surface), then insome embodiments, the interference domain may be designed to block H₂O₂not produced by the enzyme-catalyzed reaction from passing therethrough.In addition, with this particular membrane design, the interferencedomain may be configured to allow analyte and oxygen to passtherethrough with minimal impedance.

Application of the layers in forming the interference domain may beaccomplished by various methods known in the art. One coating processembodiment involves solely dip-coating and dip-rinsing steps. Anothercoating process embodiment involves solely spray-coating andspray-rinsing steps. However, a number of alternative embodimentsinvolve various use of a combination of spray-coating, dip-coating,and/or rinsing steps. For example, one dip-coating method involves thesteps of applying a coating of a first polyionic material to a substrate(e.g., the sensor or membrane layer atop the sensor, e.g., theresistance or enzyme layer) by immersing the substrate in a firstsolution of a first polyionic material; rinsing the substrate byimmersing the substrate in a rinsing solution; and, optionally, dryingthe substrate. This procedure is then repeated using a second polyionicmaterial, with the second polyionic material having charges opposite ofthe charges of the first polyionic material, in order to form apolyionic bilayer. This bilayer formation process can be repeated aplurality of times in order to produce the interference domain. In someembodiments, the number of bilayers can be from 1 to about 16 bilayers,sometimes from 1 to about 10 bilayers, and sometimes from about 3 toabout 7 bilayers. In certain embodiments, the final layer of oppositelycharged polyionic material can be deposited, such that the first and thelast layer have the same charges (both positive, or both negative). Theimmersion time for each of the coating and rinsing steps may varydepending on a number of factors. For example, immersion of thesubstrate into the polyionic solution can occur over a period of about 1to 30 minutes, or from about 2 to 20 minutes, or from about 1 to 5minutes. Rinsing may be accomplished in one step, but a plurality ofrinsing steps can also be employed. Rinsing in a series from about 2 to5 steps can be employed, with each immersion into the rinsing solutionconsuming, for example, from about 1 to about 3 minutes. In someembodiments, several polycationic solutions and/or several polyanionsolutions may be used. For example, in certain embodiments, thedip-coating sequence may involve the steps of applying a coating of afirst polycationic material to the substrate to form a first layer, thenapplying a first anionic material to the first layer to form a secondlayer, then applying a second polycationic material to the second layerto form a third layer, then applying a second polyanionic material toform a fourth layer, and then applying a first or second polycationicmaterial to the fourth layer to form a fifth layer. In some of theseembodiments, the dip-coating sequence described above may beinterspersed with rinsing steps performed between coating steps. It iscontemplated that any of a variety of permutations involving the stepsand materials described may be employed. In alternative embodiments, thematerials used to form the polycationic and/or polyanionic layers may besubstantially the same. However, the individual polycationic layers mayhave a different level of ionization than one or more other polycationiclayers in the inference domain, and the individual polyanionic layersmay also have a different level of ionization than one or more otherpolyanionic layers. For example, in one embodiment, the dip-coatingsequence method involves the use of a first solution at a first pHcomprising a polycationic material, a second solution at a second pHcomprising a polyanionic material, a third solution at a third pHcomprising the aforementioned polycationic material, a fourth solutionat a fourth pH comprising the aforementioned polyanionic material, and afifth solution at a fifth pH comprising the aforementioned polycationicmaterial. Even though the same polycationic material is used to form thefirst, third, and fifth layers, because the solution used to form thefirst, third, and fifth layers have different pHs, the ionization levelsof the first, third, and fifth layers will be different. Likewise, eventhough the same polyanionic material is used to form the second andfourth layers, because the solution used to form the second and fifthlayers have different pHs, the levels of ionization of the second andfourth layers will be different. This difference in ionization levelscan affect, inter alia, the mechanical properties of the film,structural properties (e.g., porosity, roughness) of the film,diffusional properties of the film, and also the selectivity of acertain polyelectrolyte layer for a certain interfering species overanother interfering species. All of these effects influence the abilityof the individual polyelectrolyte layers and of the interference domainto reduce transport of a variety of interfering species. In certainembodiments, at least two polycationic and/or two polyanionic layers ofthe interference domain are formed from the samepolycationic/polyanionic material, but through use of solutions atdifferent pHs. In some of these embodiments, a first polycationic layerpossesses a high selectivity for a particular interfering species overother interfering species, while a second polycationic layer possesses ahigh selectivity for a different interfering species over otherinterfering species.

Alternatively or additionally, spray coating techniques can be employed.In one embodiment, the coating process generally includes the steps ofapplying a coating of: a first polyionic material to the substrate bycontacting the substrate with a first solution of a first polyionicmaterial; rinsing the substrate by spraying the substrate with a rinsingsolution; and (optionally) drying the substrate. Similar to thedip-coating process, the spray-coating process may then be repeated witha second polyionic material, with the second polyionic material havingcharges opposite to those of the first polyionic material. Thecontacting of the substrate with solution, either polyionic material orrinsing solution, may occur through a variety of methods. For example,the substrate may be dipped into both solutions. One alternative is toapply the solutions in a spray or mist form. Of course, variouscombinations are possible and within the scope of the contemplatedembodiments, e.g., dipping the substrate in the polyionic materialfollowed by spraying the rinsing solution. The spray coating applicationmay be accomplished via a number of methods known in the art. Forexample, a conventional spray coating arrangement may be used, i.e., theliquid material is sprayed by application of fluid, which may or may notbe at an elevated or lowered pressure, through a reduced diameter nozzlewhich is directed towards the deposition target. Another spray coatingtechnique involves the use of ultrasonic energy, whereby the liquid isatomized by the ultrasonic vibrations of a spray forming tip and therebychanged to a spray.

Yet another technique involves electrostatic spray coating in which acharge is conveyed to the fluid or droplets to increase the efficiencyof the coating. A further method of atomizing liquid for spray coatinginvolves purely mechanical energy, e.g., through contacting the liquidwith a high speed reciprocating member or a high speed rotating disk.Still another method of producing microdroplets for spray coatingsinvolves the use of piezoelectric elements to atomize the liquid. Thesetechniques can be employed with air assistance or at an elevatedsolution pressure. In addition, a combination of two or more techniquesmay prove more useful with certain materials and conditions. A method ofspray application involves dispensing, with a metering pump, thepolyanion or polycation solution to an ultrasonic dispensing head. Thepolyion layer is sprayed so as to allow the surface droplets to coalesceacross the material surface. The resulting layer may then be allowed tointeract for a period of time or immediately rinsed with water or salinesolution (or other solution devoid of polyanion or polycation).

In some embodiments, the layers of the interference domain can include apolymer with a conjugated pi system. Polymers with conjugated pi systemscan contain a delocalized electron system, and can be conductive. Layersof polymers with conjugated pi systems can interact with each otherthrough intermolecular forces, such as electrostatic pi-pi interactions(i.e., pi-stacking). Conjugated polymers can provide beneficialproperties to an interference domain, such as increasing the rigidity,integrity, and/or reproducibility of the domain. In some embodiments,the polymer with a conjugated pi system can be poly acetylene,polypyrrole, polythiophene, poly(p-phenylene), poly(p-phenylenevinylene)or poly(carbazole). The interference domain can include alternatinglayers of any of the conjugated polymers mentioned above. In someembodiments, the number of layers of conjugated polymers can be from 1to about 20 layers, sometimes from about 3 to about 10 layers.

It is contemplated that in some embodiments, the thickness of theinterference domain may be from about 0.01 microns or less to about 20microns or more. In some of these embodiments, the thickness of theinterference domain may be from about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 microns to about 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 19.5 microns.In some of these embodiments, the thickness of the interference domainmay be from about 0.2, 0.4, 0.5, or 0.6, microns to about 0.8, 0.9, 1,1.5, 2, 3, or 4 microns.

Polyimine Films

In some embodiments, certain polymeric films can be used to forminterference domains. For example, certain polyimides prepared from2,2′-dimethyl-4,4′-diaminobiphenyl and the corresponding dianhydride canbe cast into films that can be employed as hydrogen peroxide-selectivemembranes. See, e.g., Ekinci et al., Turk. J. Chem. 30 (2006), 277-285.In one embodiment, a film is prepared using the following steps. First,n-methyl-2-pyrrolidene (NMP) is distilled over CaH₂ under reducedpressure and is stored over about 4 Å molecular sieves. Reagent gradepyromellitic dianhydride (PMDA) is sublimed at about 250° C. underreduced pressure and dried under vacuum at about 120° C. prior to use.The diamine is purified via recrystallization from ethanol to give shinycrystals. Next, 2,20-dimethyl-4,40-diaminobiphenyl, (about 1.06 g, about5 mmol) is dissolved in NMP (about 15 mL) in a 50 mL Schlenk tubeequipped with a nitrogen line, overhead stirrer, a xylene filledDean-Stark trap, and a condenser. PMDA (about 1.09 g, about 5 mmol) isthen added to the amine solution, followed by overnight stirringresulting in a viscous solution. After being stirred for about 3 hours,the solution is heated to reflux at about 200° C. for about 15 hours.During the polymerization process, the water generated from theimidization is allowed to distill from the reaction mixture togetherwith about 1-2 mL of xylene. After being allowed to cool to ambienttemperature, the solution is diluted with NMP and then slowly added to avigorously stirred solution of 95% ethanol. The precipitated polymer iscollected via filtration, washed with ethanol, and dried under reducedpressure at 150° C. Before coating, a substrate (e.g., Pt electrode) iscleaned and optionally polished with aqueous alumina slurry down toabout 0.05 μm. Then about 20 μL of polymer solution prepared bydissolving about 70 mg of polyimide in about 2 mL of NMP is dropped ontothe surface of the Pt electrode and allowed to dry at room temperaturefor about 3 days.

Self Assembly Techniques

A self-assembly process can be employed to build up ultrathin multilayerfilms comprising consecutively alternative anionic and cationicpolyelectrolytes on a charged surface. See, e.g., Decher et al., ThinSolid Films, 210-211 (1992) 831-835. Ionic attraction between oppositecharges is the driving force for the multilayer buildup. In contrast tochemisorption techniques that require a reaction yield of about 100% inorder to maintain surface functional density in each layer, no covalentbonds need to be formed with a self-assembly process. Additionally, anadvantage over the classic Langmuir-Blodgett technique is that asolution process is independent of the substrate size and topology.Exemplary polyelectrolytes for use in such a process include, but arenot limited to, polystyrenesulfonate sodium salt, polyvinylsulfatepotassium salt, poly-4-vinylbenzyl-(N,N-diethyl-N-methyl-)-ammoniumiodide, and poly(allylamine hydrochloride). The buildup of multilayerfilms can be conducted as follows. A solid substrate with a positivelycharged planar surface is immersed in the solution containing theanionic polyelectrolyte and a monolayer of the polyanion is adsorbed.Since the adsorption is carried out at relatively high concentrations ofpolyelectrolyte, a number of ionic groups remain exposed to theinterface with the solution and thus the surface charge is reversed.After rinsing in pure water the substrate is immersed in the solutioncontaining the cationic polyelectrolyte. Again a monolayer is adsorbedbut now the original surface charge is restored. By repeating both stepsin a cyclic fashion, alternating multilayer assemblies of both polymersare obtained. This process of multilayer formation is based on theattraction of opposite charges, and thus requires a minimum of twooppositely charged molecules. Consequently, one is able to incorporatemore than two molecules into the multilayer, simply by immersing thesubstrate in as many solutions of polyeletrolytes as desired, as long asthe charge is reversed from layer to layer. Even aperiodic multilayerassemblies can easily be prepared. In this respect, the technique ismore versatile than the Langmuir-Blodgett technique which is ratherlimited to periodically alternating layer systems. Another advantage isthat the immersion procedure does not pose principal restrictions as tothe size of the substrate or to the automation in a continuous process.

Specific examples of the preparation of such films are as follows.Polystyrenesulfonate (sodium salt, Mr=100,000) and polyvinylsulfate(potassium salt, Mr=245,000) and poly(allylamine hydrochloride),Mw=50,000-65,000) are obtained from commercial sources and employedwithout further purification.Poly-4-vinylbenzyl-(N,N-diethyl-N-methyl-)-ammonium iodide can besynthesized, as described in Decher et al., Ber. Bunsenges. Phys. Chem.,95 (1992) 1430. Alternating multilayer assemblies of all materials canbe characterized by UV/vis spectroscopy and small angle X-ray scattering(SAXS) using techniques known in the art. Direct-light microscopy andSAXS measurements can be performed with multilayer assemblies onsuitable substrates. The multilayer films can be deposited on, e.g.,atop a platinum electrode or other metal electrode, or a suitableintervening layer atop an electrode. For the adsorption of the firstlayer, an aqueous acidic solution of polystyrenesulfonate orpolyvinylsulfate can be used. Afterwards the substrate is rinsed withwater. After the adsorption of the first layer, the substrates can bestored for some weeks without noticeable deterioration of the surface.Thereafter, the cationic polyelectrolyte polyallylamine is adsorbed fromaqueous solution. In the case of the non-quarternized polyallylamine,the polycation is adsorbed from an acidic solution. All following layers(odd layer numbers) of the anionic polyelectrolytes are adsorbed fromaqueous solution. In the case of samples containing polyallylamine asthe previously adsorbed layer, polystyrenesulfonate layers can beadsorbed from an acidic solution. An adsorption time of about 20 minutesat ambient temperature can be employed, however, in certain embodimentslonger or shorter adsorption times may be acceptable. A range of polymerconcentrations (e.g., 20 to 30 mg per about 10 ml water) can provideacceptable results.

Multilayer molecular films of polyelectrolyte:calixarene andpolyelectrolyte:cyclodextrin hosts can be fabricated by alternatingadsorption of charged species in aqueous solutions onto a suitablesubstrate. See, e.g., X. Yang, Sensors and Actuators B 45 (1997) 87-92.Such a layer-by-layer molecular deposition approach can be used tointegrate molecular recognition reagents into polymer films. Thedeposition process is highly reproducible and the resulting films areuniform and stable. Replacing polyanions, highly negatively chargedmolecular species can be used for film fabrication. These molecularreagents are capable of binding organic species and can be deposited asfunctional components into thin films. This approach incorporatespolymer and molecular elements into the film and thus results in filmswith polymer's physical properties and molecular film's selectivity.Films can be prepared as follows. The substrate (e.g., Pt electrode) canbe first treated with aminopropyltrimethoxysilane in chloroform,followed with deposition of PSS and then PDDA polyelectrolytes bydipping into the aqueous solutions of the polyelectrolytes,respectively. After this, alternating depositions of negatively chargedmolecular host species (e.g., calix[6]arene or p-t-butylcalix[4]arene)and PDDA can be carried out until the desired number of bilayers isreached. Between each deposition, the substrate is thoroughly rinsedwith deionized water. The polyelectrolyte and molecular ion assembly canbe monitored by UV-vis absorption spectroscopy and mass loading can bemeasured with surface acoustic wave (SAW) devices.

In some embodiments, the interference domain is formed from one or morecellulosic derivatives. In general, cellulosic derivatives can includepolymers such as cellulose acetate, cellulose acetate butyrate,2-hydroxyethyl cellulose, cellulose acetate phthalate, cellulose acetatepropionate, cellulose acetate trimellitate, or blends and combinationsthereof.

In some alternative embodiments, other polymer types that can beutilized as a base material for the interference domain includepolyurethanes and/or polymers having controlled pore size. In one suchalternative embodiment, the interference domain includes a thin,hydrophobic membrane that is non-swellable and restricts diffusion oflow molecular weight species. The interference domain is permeable torelatively low molecular weight substances, such as hydrogen peroxide,but restricts the passage of higher molecular weight substances,including glucose and ascorbic acid. Other systems and methods forreducing or eliminating interference species that can be applied to themembrane system of the preferred embodiments are described in U.S. Pat.No. 7,074,307, U.S. Patent Publication No. US-2005-0176136-A1, U.S. Pat.No. 7,081,195, and U.S. Patent Publication No. US-2005-0143635-A1, eachof which is incorporated by reference herein in its entirety.

It is contemplated that in some embodiments, the thickness of theinterference domain can be from about 0.01 μm or less to about 20 μm ormore. In some of these embodiments, the thickness of the interferencedomain can be from about 0.01, about 0.05, about 0.1, about 0.15, about0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about0.5, about 1, about 1.5, about 2, about 2.5, about 3, or about 3.5 μmand about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, or about 19.5 μm. In some of these embodiments, thethickness of the interference domain can be from about 0.2, about 0.4,about 0.5, or about 0.6, μm to about 0.8, about 0.9, about 1, about 1.5,about 2, about 3, or about 4 μm.

In general, the membrane systems described herein can be formed ordeposited on the exposed electroactive surfaces (e.g., one or more ofthe working and reference electrodes) using known thin film techniques(for example, casting, spray coating, drawing down, electro-depositing,dip coating, and the like); however, casting or other known applicationtechniques can also be utilized. In some embodiments, the interferencedomain can be deposited by spray or dip coating. In one exemplaryembodiment, the interference domain is formed by dip coating the sensorinto an interference domain solution using an insertion rate of fromabout 0.5 inch/min to about 60 inches/min, and sometimes about 1inch/min; a dwell time of from about 0.01 minutes to about 2 minutes,and sometimes about 1 minute; and a withdrawal rate of from about 0.5inch/minute to about 60 inches/minute, and sometimes about 1inch/minute; and curing (drying) the domain from about 1 minute to about14 hours, and sometimes from about 3 minutes to about 15 minutes (andcan be accomplished at room temperature or under vacuum, e.g., about 20to about 30 mmHg). In one exemplary embodiment including a celluloseacetate butyrate interference domain, a 3-minute cure (i.e., dry) timeis used between each layer applied. In another exemplary embodimentemploying a cellulose acetate interference domain, a 15 minute cure timeis used between each layer applied.

In some embodiments, the dip process can be repeated at least one timeand up to 10 times or more. In other embodiments, only one dip ispreferred. The preferred number of repeated dip processes can dependupon the cellulosic derivative(s) used, their concentration, conditionsduring deposition (e.g., dipping) and the desired thickness (e.g.,sufficient thickness to provide functional blocking of certaininterferents), and the like. In one embodiment, an interference domainis formed from three layers of cellulose acetate butyrate. In anotherembodiment, an interference domain is formed from 10 layers of celluloseacetate. In yet another embodiment, an interference domain is formedfrom 1 layer of a blend of cellulose acetate and cellulose acetatebutyrate. In alternative embodiments, the interference domain can beformed using any known method and combination of cellulose acetate andcellulose acetate butyrate, as will be appreciated by one skilled in theart.

Curing a polymer layer deposited by solvent evaporation is sometimeshelpful to prevent sensor drift by allowing the polymer chains andhydrophilic and hydrophobic components to rearrange into a low energystate that remains stable over time. The final polymer microstructuredetermines the analyte and oxygen permeability characteristics andultimate sensor performance. Thus, an analyte sensor can be cured sothat all rearrangement occurs before the sensor is calibrated and thesensor sensitivity does not change before or during use. The speed, typeof rearrangement, and polymer layer microstructure can be controlled anddirected through exposure to different conditions, typically humidity,temperature, and time. Aqueous solvent cure can be used to acceleratepolymer rearrangement in polymer layers with mixed hydrophilic andhydrophobic domains to achieve a sensor with desirable stability,analyte permeability, and oxygen performance. Benefits over the currenthumidity and temperature curing process include the reduction in drift,a reduction in cure time, and less sensor-to-sensor variability.

Aqueous solvents for curing may contain water miscible solvents, salts,stabilizers, plasticizers, or other components to modify rearrangement.Solvent temperature can be in the range of 0° C.-100° C., morepreferably 20° C.-70° C. Miscible solvents can be alcohols or polarorganic solvents such as ethanol or THF. Salts may be of any variety,for example PBS, and can include modifying components such as biocidesand interferent species. Process may involve multiple soak steps indifferent aqueous formulations at different temperatures to selectivelyinduce particular sequential rearrangements or extract specificcompounds.

Electrode Domain

It is contemplated that in some embodiments, such as the embodimentillustrated in FIG. 2A, an optional electrode domain 42, also referredto as the electrode layer, can be provided, in addition to thebiointerface domain and the enzyme domain; however, in otherembodiments, the functionality of the electrode domain can beincorporated into the biointerface domain so as to provide a unitarydomain that includes the functionality of the biointerface domain,diffusion resistance domain, enzyme domain, and electrode domain.

In some embodiments, the electrode domain is located most proximal tothe electrochemically reactive surfaces. To facilitate electrochemicalreaction, the electrode domain can include a semipermeable coating thatmaintains hydrophilicity at the electrochemically reactive surfaces ofthe sensor interface. The electrode domain can enhance the stability ofan adjacent domain by protecting and supporting the material that makesup the adjacent domain. The electrode domain can also assist instabilizing the operation of the device by overcoming electrode start-upproblems and drifting problems caused by inadequate electrolyte. Thebuffered electrolyte solution contained in the electrode domain can alsoprotect against pH-mediated damage that can result from the formation ofa large pH gradient between the substantially hydrophobic interferencedomain and the electrodes due to the electrochemical activity of theelectrodes.

In some embodiments, the electrode domain includes a flexible,water-swellable, substantially solid gel-like film (e.g., a hydrogel)having a “dry film” thickness of from about 0.05 μm to about 100 μm, andsometimes from about 0.05, about 0.1, about 0.15, about 0.2, about 0.25,about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, or about 1 μmto about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about4.5, about 5, about 6, about 6.5, about 7, about 7.5, about 8, about8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, about 19.5, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, or about 100 μm. In some embodiments, thethickness of the electrode domain can be from about 2, about 2.5, orabout 3 μm to about 3.5, about 4, about 4.5, or about 5 μm in the caseof a transcutaneously implanted sensor, or from about 6, about 7, orabout 8 μm to about 9, about 10, about 11, or about 12 μm in the case ofa wholly implanted sensor. The term “dry film thickness” as used hereinis a broad term, and is to be given its ordinary and customary meaningto a person of ordinary skill in the art (and is not to be limited to aspecial or customized meaning), and refers without limitation to thethickness of a cured film cast from a coating formulation onto thesurface of the membrane by standard coating techniques. The coatingformulation can comprise a premix of film-forming polymers and acrosslinking agent and can be curable upon the application of moderateheat.

In certain embodiments, the electrode domain can be formed of a curablemixture of a urethane polymer and a hydrophilic polymer. In some ofthese embodiments, coatings are formed of a polyurethane polymer havinganionic carboxylate functional groups and non-ionic hydrophilicpolyether segments, which are crosslinked in the presence ofpolyvinylpyrrolidone and cured at a moderate temperature of about 50° C.

Particularly suitable for this purpose are aqueous dispersions offully-reacted colloidal polyurethane polymers having cross-linkablecarboxyl functionality (e.g., BAYBOND™; Mobay Corporation). Thesepolymers are supplied in dispersion grades having apolycarbonate-polyurethane backbone containing carboxylate groupsidentified as XW-121 and XW-123; and a polyester-polyurethane backbonecontaining carboxylate groups, identified as XW-110-2. In someembodiments, BAYBOND™123, an aqueous anionic dispersion of an aliphatepolycarbonate urethane polymer sold as a 35 wt. % solution in water andco-solvent N-methyl-2-pyrrolidone, can be used.

In some embodiments, the electrode domain is formed from a hydrophilicpolymer that renders the electrode domain equally or more hydrophilicthan an overlying domain (e.g., interference domain, enzyme domain).Such hydrophilic polymers can include, a polyamide, a polylactone, apolyimide, a polylactam, a functionalized polyamide, a functionalizedpolylactone, a functionalized polyimide, a functionalized polylactam orcombinations thereof, for example.

In some embodiments, the electrode domain is formed primarily from ahydrophilic polymer, and in some of these embodiments, the electrodedomain is formed substantially from PVP. PVP is a hydrophilicwater-soluble polymer and is available commercially in a range ofviscosity grades and average molecular weights ranging from about 18,000to about 500,000, under the PVP K™ homopolymer series by BASF Wyandotteand by GAF Corporation. In certain embodiments, a PVP homopolymer havingan average molecular weight of about 360,000 identified as PVP-K90 (BASFWyandotte) can be used to form the electrode domain. Also suitable arehydrophilic, film-forming copolymers of N-vinylpyrrolidone, such as acopolymer of N-vinylpyrrolidone and vinyl acetate, a copolymer ofN-vinylpyrrolidone, ethylmethacrylate and methacrylic acid monomers, andthe like.

In certain embodiments, the electrode domain is formed entirely from ahydrophilic polymer. Useful hydrophilic polymers contemplated include,but are not limited to, poly-N-vinylpyrrolidone,poly-N-vinyl-2-piperidone, poly-N-vinyl-2-caprolactam,poly-N-vinyl-3-methyl-2-caprolactam, poly-N-vinyl-3-methyl-2-piperidone,poly-N-vinyl-4-methyl-2-piperidone, poly-N-vinyl-4-methyl-2-caprolactam,poly-N-vinyl-3-ethyl-2-pyrrolidone,poly-N-vinyl-4,5-dimethyl-2-pyrrolidone, polyvinylimidazole,poly-N,N-dimethylacrylamide, polyvinyl alcohol, polyacrylic acid,polyethylene oxide, poly-2-ethyl-oxazoline, copolymers thereof andmixtures thereof. A blend of two or more hydrophilic polymers can bepreferred in some embodiments.

It is contemplated that in certain embodiments, the hydrophilic polymerused may not be crosslinked, but in other embodiments, crosslinking canbe used and achieved by any of a variety of methods, for example, byadding a crosslinking agent. In some embodiments, a polyurethane polymercan be crosslinked in the presence of PVP by preparing a premix of thepolymers and adding a cross-linking agent just prior to the productionof the membrane. Suitable cross-linking agents contemplated include, butare not limited to, carbodiimides (e.g.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, andUCARLNK™ XL-25 (Union Carbide)), epoxides and melamine/formaldehyderesins. Alternatively, it is also contemplated that crosslinking can beachieved by irradiation at a wavelength sufficient to promotecrosslinking between the hydrophilic polymer molecules, which isbelieved to create a more tortuous diffusion path through the domain.

The flexibility and hardness of the coating can be varied as desired byvarying the dry weight solids of the components in the coatingformulation. The term “dry weight solids” as used herein is a broadterm, and is to be given its ordinary and customary meaning to a personof ordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to the dry weightpercent based on the total coating composition after the time thecrosslinker is included. In one embodiment, a coating formulation cancontain from about 6 to about 20 dry wt. %, preferably about 8 dry wt.%, PVP; about 3 to about 10 dry wt. %, sometimes about 5 dry wt. %cross-linking agent; and from about 70 to about 91 wt. %, sometimesabout 87 wt. % of a polyurethane polymer, such as apolycarbonate-polyurethane polymer, for example. The reaction product ofsuch a coating formulation is referred to herein as a water-swellablecross-linked matrix of polyurethane and PVP.

In some embodiments, underlying the electrode domain is an electrolytephase that when hydrated is a free-fluid phase including a solutioncontaining at least one compound, typically a soluble chloride salt,which conducts electric current. In one embodiment wherein the membranesystem is used with a glucose sensor such as is described herein, theelectrolyte phase flows over the electrodes and is in contact with theelectrode domain. It is contemplated that certain embodiments can useany suitable electrolyte solution, including standard, commerciallyavailable solutions. Generally, the electrolyte phase can have the sameosmotic pressure or a lower osmotic pressure than the sample beinganalyzed. In preferred embodiments, the electrolyte phase comprisesnormal saline.

Bioactive Agents

It is contemplated that any of a variety of bioactive (therapeutic)agents can be used with the analyte sensor systems described herein,such as the analyte sensor system shown in FIG. 1. In specificembodiments, the bioactive agents can be in the biointerface layer ofthe disclosed devices. In some embodiments, the bioactive agent is ananticoagulant. The term “anticoagulant” as used herein is a broad term,and is to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to a substance theprevents coagulation (e.g., minimizes, reduces, or stops clotting ofblood). In these embodiments, the anticoagulant included in the analytesensor system can prevent coagulation within or on the sensor. Suitableanticoagulants for incorporation into the sensor system include, but arenot limited to, vitamin K antagonists (e.g., Acenocoumarol, Clorindione,Dicumarol (Dicoumarol), Diphenadione, Ethyl biscoumacetate,Phenprocoumon, Phenindione, Tioclomarol, or Warfarin), heparin groupanticoagulants (e.g. Platelet aggregation inhibitors: Antithrombin III,Bemiparin, Dalteparin, Danaparoid, Enoxaparin, Heparin, Nadroparin,Parnaparin, Reviparin, Sulodexide, Tinzaparin), other plateletaggregation inhibitors (e.g. Abciximab, Acetylsalicylic acid (Aspirin),Aloxiprin, Beraprost, Ditazole, Carbasalate calcium, Cloricromen,Clopidogrel, Dipyridamole, Epoprostenol, Eptifibatide, Indobufen,Iloprost, Picotamide, Ticlopidine, Tirofiban, Treprostinil, Triflusal),enzymes (e.g., Alteplase, Ancrod, Anistreplase, Brinase, Drotrecoginalfa, Fibrinolysin, Protein C, Reteplase, Saruplase, Streptokinase,Tenecteplase, Urokinase), direct thrombin inhibitors (e.g., Argatroban,Bivalirudin, Desirudin, Lepirudin, Melagatran, Ximelagatran, otherantithrombotics (e.g., Dabigatran, Defibrotide, Dermatan sulfate,Fondaparinux, Rivaroxaban), and the like.

In one embodiment, heparin is incorporated into the analyte sensorsystem, for example by dipping or spraying. While not wishing to bebound by theory, it is believed that heparin coated on the catheter orsensor can prevent aggregation and clotting of blood on the analytesensor system, thereby preventing thromboembolization (e.g., preventionof blood flow by the thrombus or clot) or subsequent complications. Insome embodiments, heparin is admixed with one or more zwitterioniccompounds or derivatives thereof, such as hydrolyzable cationic estersthereof (as described above), prior to dipping or spraying, thusproviding the sensor system with a mixed coating of heparin and one ormore zwitterionic compounds or derivatives thereof.

In some embodiments, an antimicrobial is coated on the catheter (inneror outer diameter) or sensor. In some embodiments, an antimicrobialagent can be incorporated into the analyte sensor system. Theantimicrobial agents contemplated can include, but are not limited to,antibiotics, antiseptics, disinfectants and synthetic moieties, andcombinations thereof, and other agents that are soluble in organicsolvents such as alcohols, ketones, ethers, aldehydes, acetonitrile,acetic acid, methylene chloride and chloroform. The amount of eachantimicrobial agent used to impregnate the medical device varies to someextent, but is at least of an effective concentration to inhibit thegrowth of bacterial and fungal organisms, such as staphylococci,gram-positive bacteria, gram-negative bacilli and Candida.

In some embodiments, an antibiotic can be incorporated into the analytesensor system. Classes of antibiotics that can be used includetetracyclines (e.g., minocycline), rifamycins (e.g., rifampin),macrolides (e.g., erythromycin), penicillins (e.g., nafeillin),cephalosporins (e.g., cefazolin), other β-lactam antibiotics (e.g.,imipenem, aztreonam), aminoglycosides (e.g., gentamicin),chloramphenicol, sulfonamides (e.g., sulfamethoxazole), glycopeptides(e.g., vancomycin), quinolones (e.g., ciprofloxacin), fusidic acid,trimethoprim, metronidazole, clindamycin, mupirocin, polyenes (e.g.,amphotericin B), azoles (e.g., fluconazole), and β-lactam inhibitors(e.g., sulbactam).

Examples of specific antibiotics that can be used include minocycline,rifampin, erythromycin, nafcillin, cefazolin, imipenem, aztreonam,gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim,metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin,clarithromycin, ofloxacin, lomefloxacin, norfloxacin, nalidixic acid,sparfloxacin, pefloxacin, amifloxacin, enoxacin, fleroxacin,temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid,amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin.

In some embodiments, an antiseptic or disinfectant can be incorporatedinto the analyte sensor system. Examples of antiseptics anddisinfectants are hexachlorophene, cationic bisiguanides (e.g.,chlorhexidine, cyclohexidine) iodine and iodophores (e.g.,povidoneiodine), para-chloro-meta-xylenol, triclosan, furan medicalpreparations (e.g., nitrofurantoin, nitrofurazone), methenamine,aldehydes (glutaraldehyde, formaldehyde) and alcohols. Other examples ofantiseptics and disinfectants will readily suggest themselves to thoseof ordinary skill in the art.

In some embodiments, an anti-barrier cell agent can be incorporated intothe analyte sensor system. Anti-barrier cell agents can includecompounds exhibiting effects on macrophages and foreign body giant cells(FBGCs). It is believed that anti-barrier cell agents prevent closure ofthe barrier to solute transport presented by macrophages and FBGCs atthe device-tissue interface during FBC maturation. Anti-barrier cellagents can provide anti-inflammatory or immunosuppressive mechanismsthat affect the wound healing process, for example, healing of the woundcreated by the incision into which an implantable device is inserted.Cyclosporine, which stimulates very high levels of neovascularizationaround biomaterials, can be incorporated into a biointerface membrane ofa preferred embodiment (see U.S. Pat. No. 5,569,462 to Martinson etal.). Alternatively, Dexamethasone, which abates the intensity of theFBC response at the tissue-device interface, can be incorporated into abiointerface membrane of a preferred embodiment. Alternatively,Rapamycin, which is a potent specific inhibitor of some macrophageinflammatory functions, can be incorporated into a biointerface membraneof a preferred embodiment.

In some embodiments, an anti-inflammatory agent can be incorporated intothe analyte sensor system to reduce acute or chronic inflammationadjacent to the implant or to decrease the formation of a FBC capsule toreduce or prevent barrier cell layer formation, for example. Suitableanti-inflammatory agents include but are not limited to, for example,nonsteroidal anti-inflammatory drugs (NSAIDS) such as acetometaphen,aminosalicylic acid, aspirin, celecoxib, choline magnesiumtrisalicylate, diclofenac potassium, diclofenac sodium, diflunisal,etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, interleukin(IL)-10, IL-6 mutein, anti-IL-6 iNOS inhibitors (for example, L-NAME orL-NMDA), Interferon, ketoprofen, ketorolac, leflunomide, melenamic acid,mycophenolic acid, mizoribine, nabumetone, naproxen, naproxen sodium,oxaprozin, piroxicam, rofecoxib, salsalate, sulindac, and tolmetin; andcorticosteroids such as cortisone, hydrocortisone, methylprednisolone,prednisone, prednisolone, betamethesone, beclomethasone dipropionate,budesonide, dexamethasone sodium phosphate, flunisolide, fluticasonepropionate, paclitaxel, tacrolimus, tranilast, triamcinolone acetonide,betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate,betamethasone valerate, desonide, desoximetasone, fluocinolone,triamcinolone, triamcinolone acetonide, clobetasol propionate, anddexamethasone.

In some embodiments, an immunosuppressive or immunomodulatory agent canbe incorporated into the analyte sensor system in order to interferedirectly with several key mechanisms necessary for involvement ofdifferent cellular elements in the inflammatory response. Suitableimmunosuppressive and immunomodulatory agents include, but are notlimited to, anti-proliferative, cell-cycle inhibitors, (for example,paclitaxel, cytochalasin D, infiximab), taxol, actinomycin, mitomycin,thospromote VEGF, estradiols, NO donors, QP-2, tacrolimus, tranilast,actinomycin, everolimus, methothrexate, mycophenolic acid, angiopeptin,vincristing, mitomycine, statins, C MYC antisense, sirolimus (andanalogs), RestenASE, 2-chloro-deoxyadenosine, PCNA Ribozyme, batimstat,prolyl hydroxylase inhibitors, PPARy ligands (for example troglitazone,rosiglitazone, pioglitazone), halofuginone, C-proteinase inhibitors,probucol, BCP671, EPC antibodies, catchins, glycating agents, endothelininhibitors (for example, Ambrisentan, Tesosentan, Bosentan), Statins(for example, Cerivasttin), E. coli heat-labile enterotoxin, andadvanced coatings.

In some embodiments, an anti-infective agent can be incorporated intothe analyte sensor system. In general, anti-infective agents aresubstances capable of acting against infection by inhibiting the spreadof an infectious agent or by killing the infectious agent outright,which can serve to reduce an immuno-response without an inflammatoryresponse at the implant site, for example. Anti-infective agentsinclude, but are not limited to, anthelmintics (e.g., mebendazole),antibiotics (e.g., aminoclycosides, gentamicin, neomycin, tobramycin),antifungal antibiotics (e.g., amphotericin b, fluconazole, griseofulvin,itraconazole, ketoconazole, nystatin, micatin, tolnaftate),cephalosporins (e.g., cefaclor, cefazolin, cefotaxime, ceftazidime,ceftriaxone, cefuroxime, cephalexin), β-lactam antibiotics (e.g.,cefotetan, meropenem), chloramphenicol, macrolides (e.g., azithromycin,clarithromycin, erythromycin), penicillins (e.g., penicillin G sodiumsalt, amoxicillin, ampicillin, dicloxacillin, nafcillin, piperacillin,ticarcillin), tetracyclines (e.g., doxycycline, minocycline,tetracycline), bacitracin, clindamycin, colistimethate sodium, polymyxinb sulfate, vancomycin, antivirals (e.g., acyclovir, amantadine,didanosine, efavirenz, foscarnet, ganciclovir, indinavir, lamivudine,nelfinavir, ritonavir, saquinavir, silver, stavudine, valacyclovir,valganciclovir, zidovudine), quinolones (e.g., ciprofloxacin,levofloxacin); sulfonamides (e.g., sulfadiazine, sulfisoxazole),sulfones (e.g., dapsone), furazolidone, metronidazole, pentamidine,sulfanilamidum crystallinum, gatifloxacin, andsulfamethoxazole/trimethoprim.

In some embodiments, a vascularization agent can be incorporated intothe analyte sensor system. Vascularization agents generally can includesubstances with direct or indirect angiogenic properties. In some cases,vascularization agents can additionally affect formation of barriercells in vivo. By indirect angiogenesis, it is meant that theangiogenesis can be mediated through inflammatory or immune stimulatorypathways. It is not fully known how agents that induce localvascularization indirectly inhibit barrier-cell formation; however,while not wishing to be bound by theory, it is believed that somebarrier-cell effects can result indirectly from the effects ofvascularization agents.

Vascularization agents can provide mechanisms that promoteneovascularization and accelerate wound healing around the membrane orminimize periods of ischemia by increasing vascularization close to thetissue-device interface. Sphingosine-1-Phosphate (S1P), a phospholipidpossessing potent angiogenic activity, can be incorporated into thebiointerface membrane. Monobutyrin, a vasodilator and angiogenic lipidproduct of adipocytes, can also be incorporated into the biointerfacemembrane. In another embodiment, an anti-sense molecule (for example,thrombospondin-2 anti-sense), which can increase vascularization, isincorporated into a biointerface membrane.

Vascularization agents can provide mechanisms that promote inflammation,which is believed to cause accelerated neovascularization and woundhealing in vivo. In one embodiment, a xenogenic carrier, for example,bovine collagen, which by its foreign nature invokes an immune response,stimulates neovascularization, and is incorporated into a biointerfacemembrane of some embodiments. In another embodiment, Lipopolysaccharide,an immunostimulant, can be incorporated into a biointerface membrane. Inanother embodiment, a protein, for example, a bone morphogenetic protein(BMP), which modulates bone healing in tissue, can be incorporated intothe biointerface membrane.

In some embodiments, an angiogenic agent can be incorporated into theanalyte sensor system. Angiogenic agents are substances capable ofstimulating neovascularization, which can accelerate and sustain thedevelopment of a vascularized tissue bed at the tissue-device interface,for example. Angiogenic agents include, but are not limited to, BasicFibroblast Growth Factor (bFGF), (also known as Heparin Binding GrowthFactor-II and Fibroblast Growth Factor II), Acidic Fibroblast GrowthFactor (aFGF), (also known as Heparin Binding Growth Factor-I andFibroblast Growth Factor-I), Vascular Endothelial Growth Factor (VEGF),Platelet Derived Endothelial Cell Growth Factor BB (PDEGF-BB),Angiopoietin-1, Transforming Growth Factor Beta (TGF-β), TransformingGrowth Factor Alpha (TGFα), Hepatocyte Growth Factor, Tumor NecrosisFactor-Alpha (TNFα), Placental Growth Factor (PLGF), Angiogenin,Interleukin-8 (IL-8), Hypoxia Inducible Factor-I (HIF-1),Angiotensin-Converting Enzyme (ACE) Inhibitor Quinaprilat, Angiotropin,Thrombospondin, Peptide KGHK, Low Oxygen Tension, Lactic Acid, Insulin,Copper Sulphate, Estradiol, prostaglandins, cox inhibitors, endothelialcell binding agents (e.g., decorin or vimentin), glenipin, hydrogenperoxide, nicotine, and Growth Hormone.

In some embodiments, a pro-inflammatory agent can be incorporated intothe analyte sensor system. Pro-inflammatory agents are generallysubstances capable of stimulating an immune response in host tissue,which can accelerate or sustain formation of a mature vascularizedtissue bed. For example, pro-inflammatory agents are generally irritantsor other substances that induce chronic inflammation and chronicgranular response at the wound-site. While not wishing to be bound bytheory, it is believed that formation of high tissue granulation inducesblood vessels, which supply an adequate or rich supply of analytes tothe device-tissue interface. Pro-inflammatory agents include, but arenot limited to, xenogenic carriers, Lipopolysaccharides, S. aureuspeptidoglycan, and proteins.

These bioactive agents can be used alone or in combination. Thebioactive agents can be dispersed throughout the material of the sensor,for example, incorporated into at least a portion of the membranesystem, or incorporated into the device (e.g., housing) and adapted todiffuse through the membrane.

There are a variety of systems and methods by which a bioactive agentcan be incorporated into the sensor membrane. In some embodiments, thebioactive agent can be incorporated at the time of manufacture of themembrane system. For example, the bioactive agent can be blended priorto curing the membrane system, or subsequent to membrane systemmanufacture, for example, by coating, imbibing, solvent-casting, orsorption of the bioactive agent into the membrane system. Although insome embodiments the bioactive agent is incorporated into the membranesystem, in other embodiments the bioactive agent can be administeredconcurrently with, prior to, or after insertion of the device in vivo,for example, by oral administration, or locally, by subcutaneousinjection near the implantation site. A combination of bioactive agentincorporated in the membrane system and bioactive agent administrationlocally or systemically can be preferred in certain embodiments.

In general, a bioactive agent can be incorporated into the membranesystem, or incorporated into the device and adapted to diffusetherefrom, in order to modify the in vivo response of the host to themembrane. In some embodiments, the bioactive agent can be incorporatedonly into a portion of the membrane system adjacent to the sensingregion of the device, over the entire surface of the device except overthe sensing region, or any combination thereof, which can be helpful incontrolling different mechanisms or stages of in vivo response (e.g.,thrombus formation). In some alternative embodiments however, thebioactive agent can be incorporated into the device proximal to themembrane system, such that the bioactive agent diffuses through themembrane system to the host circulatory system.

The bioactive agent can include a carrier matrix, wherein the matrixincludes one or more of collagen, a particulate matrix, a resorbable ornon-resorbable matrix, a controlled-release matrix, or a gel. In someembodiments, the carrier matrix includes a reservoir, wherein abioactive agent is encapsulated within a microcapsule. The carriermatrix can include a system in which a bioactive agent is physicallyentrapped within a polymer network. In some embodiments, the bioactiveagent is cross-linked with the membrane system, while in others thebioactive agent is sorbed into the membrane system, for example, byadsorption, absorption, or imbibing. The bioactive agent can bedeposited in or on the membrane system, for example, by coating,filling, or solvent casting. In certain embodiments, ionic and nonionicsurfactants, detergents, micelles, emulsifiers, demulsifiers,stabilizers, aqueous and oleaginous carriers, solvents, preservatives,antioxidants, or buffering agents are used to incorporate the bioactiveagent into the membrane system.

In some embodiments, the surface of the membrane system comprises a tielayer found on the outermost surface of the sensor membrane to which thebioactive agent reversibly binds. In some embodiments, this tie layercomprises one or more zwitterionic compounds, or precursors orderivatives thereof, which are bound to surface-active groups of thepolymer comprising the outermost domain of the membrane system. In someembodiments, the zwitterionic compounds or precursors or derivativesthereof comprise one or more zwitterionic betaines, as described above.In some embodiments, the zwitterionic compounds or precursors orderivatives thereof comprise hydrolyzable cationic esters of azwitterionic compound, as described above. In preferred embodiments, thetie layer comprises one or more hydrolyzable cationic betaine esters,such as hydrolyzable cationic pCB esters.

The bioactive agent also can be incorporated into a polymer usingtechniques such as described above, and the polymer can be used to formthe membrane system, coatings on the membrane system, portions of themembrane system, or any portion of the sensor system.

The membrane system can be manufactured using techniques known in theart. The bioactive agent can be sorbed into the membrane system, forexample, by soaking the membrane system for a length of time (forexample, from about an hour or less to about a week, or more preferablyfrom about 4, about 8, about 12, about 16, or about 20 hours to about 1,about 2, about 3, about 4, about 5, or about 7 days).

The bioactive agent can be blended into uncured polymer prior to formingthe membrane system. The membrane system is then cured and the bioactiveagent thereby cross-linked or encapsulated within the polymer that formsthe membrane system.

In yet another embodiment, microspheres are used to encapsulate thebioactive agent. The microspheres can be formed of biodegradablepolymers, most preferably synthetic polymers or natural polymers such asproteins and polysaccharides. As used herein, the term polymer is usedto refer to both to synthetic polymers and proteins. U.S. Pat. No.6,281,015 discloses some systems and methods that can be used inconjunction with the preferred embodiments. In general, bioactive agentscan be incorporated in (1) the polymer matrix forming the microspheres,(2) microparticle(s) surrounded by the polymer which forms themicrospheres, (3) a polymer core within a protein microsphere, (4) apolymer coating around a polymer microsphere, (5) mixed in withmicrospheres aggregated into a larger form, or (6) a combinationthereof. Bioactive agents can be incorporated as particulates or byco-dissolving the factors with the polymer. Stabilizers can beincorporated by addition of the stabilizers to the factor solution priorto formation of the microspheres.

The bioactive agent can be incorporated into a hydrogel and coated orotherwise deposited in or on the membrane system. Some hydrogelssuitable for use in the preferred embodiments include cross-linked,hydrophilic, three-dimensional polymer networks that are highlypermeable to the bioactive agent and are triggered to release thebioactive agent based on a stimulus.

The bioactive agent can be incorporated into the membrane system bysolvent casting, wherein a solution including dissolved bioactive agentis disposed on the surface of the membrane system, after which thesolvent is removed to form a coating on the membrane surface.

The bioactive agent can be compounded into a plug of material, which isplaced within the device, such as is described in U.S. Pat. Nos.4,506,680 and 5,282,844, which are incorporated by reference herein intheir entireties. In some embodiments, it is preferred to dispose theplug beneath a membrane system; in this way, the bioactive agent iscontrolled by diffusion through the membrane, which provides a mechanismfor sustained-release of the bioactive agent in the host.

Release of Bioactive Agents

Numerous variables can affect the pharmacokinetics of bioactive agentrelease. The bioactive agents of the disclosed embodiments can beoptimized for short- or long-term release. In some embodiments, thebioactive agents of the disclosed embodiments are designed to aid orovercome factors associated with short-term effects (e.g., acuteinflammation or thrombosis) of sensor insertion. In some embodiments,the bioactive agents are designed to aid or overcome factors associatedwith long-term effects, for example, chronic inflammation or build-up offibrotic tissue or plaque material. In some embodiments, the bioactiveagents combine short- and long-term release to exploit the benefits ofboth.

As used herein, “controlled,” “sustained,” or “extended” release of thefactors can be continuous or discontinuous, linear or non-linear. Thiscan be accomplished using one or more types of polymer compositions,drug loadings, selections of excipients or degradation enhancers, orother modifications, administered alone, in combination or sequentiallyto produce the desired effect.

Short-term release of the bioactive agent in the disclosed embodimentsgenerally refers to release over a period of from about a few minutes orhours to about 2, about 3, about 4, about 5, about 6, or about 7 days ormore.

Loading of Bioactive Agents

The amount of loading of the bioactive agent into the membrane systemcan depend upon several factors. For example, the bioactive agent dosageand duration can vary with the intended use of the membrane system, forexample, the intended length of use of the device and the like;differences among patients in the effective dose of bioactive agent;location and methods of loading the bioactive agent; and release ratesassociated with bioactive agents and optionally their carrier matrix.Therefore, one skilled in the art will appreciate the variability in thelevels of loading the bioactive agent, for the reasons described above.

In some embodiments, in which the bioactive agent is incorporated intothe membrane system without a carrier matrix, the preferred level ofloading of the bioactive agent into the membrane system can varydepending upon the nature of the bioactive agent. The level of loadingof the bioactive agent is preferably sufficiently high such that abiological effect (e.g., thrombosis prevention) is observed. Above thisthreshold, the bioactive agent can be loaded into the membrane system soas to imbibe up to 100% of the solid portions, cover all accessiblesurfaces of the membrane, or fill up to 100% of the accessible cavityspace. Typically, the level of loading (based on the weight of bioactiveagent(s), membrane system, and other substances present) is from about 1ppm or less to about 1000 ppm or more, preferably from about 2, about 3,about 4, or about 5 ppm up to about 10, about 25, about 50, about 75,about 100, about 200, about 300, about 400, about 500, about 600, about700, about 800, or about 900 ppm. In certain embodiments, the level ofloading can be about 1 wt. % or less up to about 50 wt. % or more,preferably from about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 15, or about 20 wt. % up to about 25,about 30, about 35, about 40, or about 45 wt. %.

When the bioactive agent is incorporated into the membrane system with acarrier matrix, such as a gel, the gel concentration can be optimized,for example, loaded with one or more test loadings of the bioactiveagent. It is generally preferred that the gel contain from about 0.1 orless to about 50 wt. % or more of the bioactive agent(s), preferablyfrom about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7,about 0.8, or about 0.9 wt. % to about 6, about 7, about 8, about 9,about 10, about 15, about 20, about 25, about 30, about 35, about 40, orabout 45 wt. % or more bioactive agent(s), more preferably from about 1,about 2, or about 3 wt. % to about 4 or about 5 wt. % of the bioactiveagent(s). Substances that are not bioactive can also be incorporatedinto the matrix.

Referring now to microencapsulated bioactive agents, the release of theagents from these polymeric systems generally occurs by two differentmechanisms. The bioactive agent can be released by diffusion throughaqueous filled channels generated in the dosage form by the dissolutionof the agent or by voids created by the removal of the polymer solventor a pore forming agent during the original micro-encapsulation.Alternatively, release can be enhanced due to the degradation of theencapsulating polymer. With time, the polymer erodes and generatesincreased porosity and microstructure within the device. This createsadditional pathways for release of the bioactive agent.

In some embodiments, the sensor is designed to be bioinert, e.g., by theuse of bioinert materials. Bioinert materials do not substantially causeany response from the host. As a result, cells can live adjacent to thematerial but do not form a bond with it. Bioinert materials include butare not limited to alumina, zirconia, titanium oxide or other bioinertmaterials generally used in the “catheter/catheterization” art. Whilenot wishing to be bound by theory, it is believed that inclusion of abioinert material in or on the sensor can reduce attachment of bloodcells or proteins to the sensor, thrombosis or other host reactions tothe sensor.

EXAMPLES Example 1 Biointerface Polymers and Characterization

Various biointerface polymers were prepared using different amounts ofhard segments, PEG, and sulfobetaines. The hard segments (HS) werepolyurethanes or polyureas prepared from diisocyanates reacted with diolor diamine chain extenders of less than 12 carbon units. The particularformulations are shown in Table 1.

TABLE 1 PEG Betaine Name (wt. %) (wt. %) HS (wt. %) Mn (Da) PDI (Mw/Mn)SBL-3 20 18 35 107,000 1.7 SBL-10 35 40 25 127,000 1.6 SBL-1 21 0 3561,000 1.6 SBL-9 27 34 31 77,400 1.7 SBL-8 23 27 35 140,000 2.1 RL-7* 00 42 45,000 1.7 *Comparative.

Example 2 Characterization Analysis

Sensors were built as described in the section entitled “sensorsystems.” The in vitro response time was tested and compared to a sensorwithout the biointerface layer. It was found that the sensor with thebiointerface layer had the same T95 response time as the sensor withoutthe biointerface layer, indicating that the biointerface layer did notslow the response times of a glucose sensor (FIG. 5)

The in vivo response time was also tested in pig over the course of 15days. In particular continuous sensors with SBL-3 and crosslinked SBL-9biointerface layers were compared to a sensor without a biointerfacelayers. It was likewise found that there was no difference in responsetimes for the sensors with and without the biointerface layer (FIG. 6).This again showed that the biointerface domain did not affect theglucose sensor response time.

The cal-check performance of sensors without a biointerface layer andsensors dip coated with 5 wt. % SBL-8 were compared. The results areshown in FIG. 7. In this cal-check test, the following characteristicswere measured:

-   -   i. Glucose Slope (pA/mg/dL)—Ordinary least-squares linear        regression analysis of the electrical response of the sensor        when placed in buffer solutions of increasing glucose        concentration. It is also referred to as glucose sensitivity.    -   ii. Baseline Equivalent (mg/dL)—mg/dL equivalent of the        non-glucose related signal.    -   iii. MARD (%)—Mean Absolute Relative Difference, the measure of        variation away from the ideal line.    -   iv. Low Oxygen Response—Defined as the percent change in        electrical response under reduced oxygen conditions (i.e., at        0.25±0.05 mg O₂/L) compared with signal obtained under        atmospheric conditions. It is also referred to as Oxygen        Performance.    -   v. Acetaminophen Bias—the mg/dL equivalent signal from a 2 mg/dL        concentration of acetaminophen. Also referred to as glucose        equivalence.

The mechanical strength was also visually inspected by viewing sensorswith BL-8, a blend of SBL-8 and SBL-10, and SBL-10. The BL-10 layer hadvarious inclusions, which suggests that as the hydrophilicity of thelayer increases, the mechanical strength decreases (FIG. 8). Thehydrophilicity could also be adjusted by blending bionterface layerswith different hydrophilicities (e.g., blending a polymer that is morehydrophilic with one that is less hydrophilic to arrive at a desiredhydrophilicity and strength).

The hydrophilicity of various biointerface layers were tested bymeasuring the wt. % of water they absorbed. Crosslinked SBL-10(XSBL-10), with sulfobetaines in the backbone, was the most hydrophilicpolymer tested (FIG. 9). These data indicate that the disclosedbiointerface polymers are very hydrophilic, much more so than currentsensors without such layers. Further, one can use crosslinking to affectthe hydrophilicity of the polymer.

The effect crosslinking had on the rate water is absorbed and thetensile strength was also tested. Specifically, crosslinked (XSBL-8) anduncrosslinked (SBL-8) with sulfobetaines in the backbone were soaked inwater over time and weighed. The uncrosslinked polymer absorbed morewater over time. The crosslinked polymer absorbed less water and reachedequilibrium quicker. The crosslinked polymer also had significantly morestrength (FIG. 10A and FIG. 10B).

Hydrophobicity was also measured by contact angle experiments.Specifically, the advancing contact angles were measured using AttensionSigma force tensiometer 701 (Biolin Scientific product) on sensor wirecoated with different polymer coatings. The sensors were submerged indeionized water at 25° C. and allowed to retract and advance repeatedlyand the average values of advancing contact angles were calculated. Themore hydrophilic and wettable surface the coating is, the lower valuewould be. The baseline value for sensors without a biointerface layerwas 60°. The contact angle of the biointerface layers tested ranged fromabout 50 to about 90° (FIG. 18).

The cure rate of various crosslinkers were tested and the data is shownin FIG. 11. The curing rate is measured by the time to reach 50% ofconversion of the functional group such as isocyanate groups. Inpractice, the fast curing rate can help improve conversion andaccelerate the process however may compromise the pot-life. It isdesirable to select a curing chemistry and crosslinker that yieldsbalanced pot-life and curing rate. This can be achieve by screeningdifferent type of crosslinker and select the one giving optimumproperties.

Films of SBL-3, CBL-8, and resistance layer (i.e., a membrane in whichthere was no biointerface layer and the resistance layer formed theoutermost layer) were prepared by casting polymer solutions ontopolycarbonate sheets and a draw bar coater to cast flat films. Thisprocess was repeated until dried films were at desired thickness (3-4thou). Films were then cut into dog-bones using a dog-bone cutter and ahand press. Dog-bone films were subject to tensile test using an Instron3345 and stress-strain curves were generated. Five dog-bones weremeasured for each sample and data were presented by using average withstandard deviation as error bar. Ultimate tensile strength, tensilestrain and max load, and Young's modulus at 100% extension werecalculated from these curves. The ultimate tensile strength of SBL-3 ishigher than that of CBL-8 and the resistance layer film (FIG. 28). Thetensile strain at maximum load for SBL-3 is higher than CBL-8 and theresistance layer film (FIG. 29). The tensile strain at maximum load forCBL-8 is significantly lower than that of SBL-3 (600% vs. 400%). TheYoung's modulus at 100% extension follows a similar trend as ultimatetensile strength, with the SBL-3 having significantly higher Young'smodulus compared to both CBL-8 and resistance layer control (FIG. 30).

In terms of bulk mechanical properties, SBL-3 has higher ultimatetensile strength, tensile train at break and Young's modulus than CBL-8.In terms of solvent pairs used in polymer synthesis, SBL-3 made withTHF/EtOH has stronger mechanical properties than SBL-3 made with THF/IPAsolvent pair. In addition, dry SBL-3 with high solution viscosity (>90cps in THF/EtOH at 10.5 wt % solid content) demonstrated improved filmmechanical properties than the resistance layer control.

Example 3 Antifouling Properties

The mechanisms of protein adsorption and antifouling properties of thebiointerface layer were explored. The use of zwitterions embedded withinand physically, rather than covalently, contained in a polymer arebelieved to work via the migration of these hydrophilic species to theinterface between the polymer and the environment. Biointerface layermade from SBL-10 was tested by X-ray photoelectron spectroscopy (XPS) ina dry state and after soaking. It was found that the atomicconcentration of Sulfur on the SBL-10 coated surface of sensor tip didnot increase before soaking (dry state, 0.3%) and after soaking (0.2%),which indicate there was no migration to the surface of the zwitterionicsegments in the polymer chain (FIG. 13). Thus, while not wishing to bebound by theory, it is believed that the biointerface layer creates aloosely bonded water layer at the surface which prevents the adsorptionof proteins and cells (FIG. 12).

As another theory for the antifouling characteristics of the disclosedbiointerface layers, the swelling ability of the biointerface layer. Theability of the disclosed biointerface layer to swell from 50 to 400% isillustrated in FIG. 16. While not wishing to be bound by theory, theability to swell at the site of implant is believed to fill in voidspaces, helping exclude cells, proteins, and cytokines from the citethat may contribute to fouling.

The amount of protein adsorption was determined for a continuous glucosesensor having no biointerface layer, a continuous glucose sensor havinga polyurethaneurea with betaines in the main polymer chain, and acontinuous glucose sensor having polyurethaneurea with betaines at onlythe ends of the polymer chain. Fluorescently conjugated constructs ofbovine serum albumin and human fibrinogen were incubated with thesesensor configurations for lhr. These sensors were then washed in DPBSand imaged as a z-stack on a laser scanning confocal. Maximum intensityprojections were made of this image stack and the fluorescentintensities of protein adsorbed on the outer membrane above the sensoractive electrode region was measured and the results (a.u.) are shown inFIG. 19. A similar test was performed using polymers SBL-1, SBL-3, andSBL-10, as compared to a silicone polycarbonate urethane layer (RL-1)and a silicone-free polycarbonate urethane layer (RL-2) (see FIG. 20).The data show that having the betaine groups within the polymer chainresulted in significantly less protein adsorption than when the betainegroups are on the ends of the polymer chains.

The protein results were confirmed using an in vitro on sensor assayusing the micro-bca extraction method. Sensors were placed into humanplasma for lhr. The protein adsorbed on the surface was extracted with adetergent solution and was tested for total protein using the micro-BCA(bicinchoninic acid assay). Again it was found that the biointerfacepolymers SBL-3 and SBL-10 resulted in significant reductions in totalprotein adsorption as compared to sensors without a biointerface layer(FIG. 21).

Normalized protein adsorption of BSA or fibrinogen for sensors without abiointerface layer (RL-1 and RL-2), a sensor without betaines or PEG inthe backbone (RL-7), and sensors with biointerface layers SBL-3,crosslinked versions of SBL-10, SBL-8, and SBL-9 (XSBL-10, XSL-8, andXSL-9, respectively), and crosslinked and uncrosslinked versions ofSBL-3 where the sulfobetaines have been replaced with carboxylbetains(XCBL-3 and CBL-3, respectively). The significant reduction in proteinadsorption with the biointerface layers is evident (FIG. 22). Z-stackimages of certain polymers were taken in skive region of these sensors(FIG. 31). Both SBL-3 and CBL-8 performed very well and have minimalprotein adsorption to their surfaces (indicated in green) compared toboth RL-7 (non-betaine control) and a resistance layer. FIG. 32 providesthe quantification results based on images in FIG. 31.

Both carboxyl and sulfo-betaine type coatings evaluated on spin-coatedglass discs and dip coated onto sensors had protein adsorption that wassignificantly less than both non-betaine and non-coated RL controls.

The thicknesses of fibrous capsules were also measured. Fibrous capsulesare a step in the biomaterial-associated inflammation response. It wasfound that the thickness of fibrous capsules was approximately 26% lesswhen using a biointerface polymer (SBL-3) versus no biointerface layer(FIG. 23).

The stability of the biointerface layer after 14 days in vivo wasvisually confirmed. The layer before implant can be compared to SBL-3(middle-row) and SBL-10 (bottom row). The SBL-10 showed more degradationthan SBL-3 (FIG. 24).

The raw counts of data from continuous glucose sensors were plotted atday 2 and day 14. It was found that without the biointerface layer,there was a greater difference between sensors at day 14 than withsensors that had the biointerface layer. This data suggests thatbiofouling was a more significant factor, leading to more variance inthe measurements, when no biointerface layer is used (FIG. 25).

Example 4 Noise Analysis

An ambulatory pig model was developed for the in vivo assessment ofsensor performance. Using hair-less Yucatan pigs, sensors and wearablepods were adhered to their skin, which is similar to humans. Two 10french external vascular access ports that allow infusion and withdrawalof fluids and blood from central venous circulation were installed intothe descending right/left jugular veins. Animals were allowed to recoverfor 5-7 days before study. Pigs were induced with 5% isoflurane usingSurgivet anesthesia machine for 5-10 minutes. Pigs were maintained at2.5%-4% isoflurane during the duration of the sensor insertion. A pulseoximeter was used to track pig telemetry and pO2. The pigs' skin wascleaned using soap and chlorohexidine surgical scrub. The pigs' skin wasthen cleaned with alcohol gauze and then prepared with skin tac andallowed to air dry. The sensor was inserted and active transmitter inlogging mode was snapped in. Animal patch overlays were used to securethe patch to the skin for long duration wear. Tegaderm (4″ inch wide)was used to protect patch edges from lifting and waterproofing allsensors.

A 25% dextrose bag was prepared from 0.9% saline bag mixed (1000 mL)with 50% dextrose. Baseline measurements were taken from sampling bloodfrom the vascular access ports. The 25% dextrose bag was hooked up toinfuse the vascular access port and started at a basal drip rate of1-1.5 drops per second. Measurements were taken every 10 minutes for theduration of the excursion. Data was downloaded from transmitters anddata was processed in MATLAB for time lag, sensitivity, noise, EOL, andMARD. There was minimal differences between groups in terms of time lagimprovement, slope stability, and MARD calculation. Thus thebiointerface layers do not affect cal-check or drift profiles. FurtherSBL-3 exhibits lowest levels of noise compared to CBL-8 and SBL-3 groups(FIG. 27). There are no apparent detriments in performance betweengroups in terms of time lag, slope, or MARD.

Example 5 Fluorescent Incorporation

A fluorescent dye labeled polyurethaneurea (FPUU) was synthesized by twostep polycondensation reaction using erythrosine B dye (0.21 wt. %). TheFPUU can be made in ethyl acetate and isopropanol mixed solvent and formhomogeneous transparent red solution. The polymer can be precipitated inhexane. The polymer precipitation has strong bright red color, while thesupernatant hexane is colorless transparent, which indicate that all thedye was covalently incorporated into polymer. After soaking the polymersin water for one week, no free dye leached out.

Example 6 Synthesis of SBL-8

The biointerface polymer is a polyurethaneurea that was synthesized viaa two-step polycondensation reaction. In the first step, a homogeneouspolyurethane prepolymer with isocyanate end groups on both prepolymerchain ends was prepared. In the second step, a small molecular diaminewere used as chain extenders. These diamines react with prepolymer indilute solution to obtain well-defined polyurethaneurea with highmolecular weight.

Using SBL-8 as a representative example, the prepolymer was prepared byadding isophorone diisocyanate (IPDI), polyethylene oxide diol (YMER™N120), polycarbonate diol (Ravecarb 107 polycarbonate diol), andsulfobetaine prepolymer into a dry 200 mL reaction jar fitted with anitrogen sparge tube and a mechanical stirrer at room temperature. Thereaction was heated to 65° C. for 30 min under nitrogen with mechanicalstirring (200 rpm) until all the reactants dissolved. 400 ppm ofcatalyst were added into the reaction, which was kept at 65° C. for 1 h.The reaction temperature was raised to 85° C. and kept for 3 h until nobubbles were observed in the reaction mixture. The reaction mixture wasallowed to stir for an additional 2 h at 100° C. to complete theformation of the prepolymer. The viscous prepolymer was cooled to 50° C.and dissolved in ethyl acetate to form a transparent solution.

For the chain extension step, isophorone diamines were used as chainextenders and were added to a dry 700 mL reaction jar equipped with amechanical stirrer and diluted with ethyl acetate/isopropanol solventmixture. The polyurethane prepolymer solution was added dropwise intothe chain extender solution at room temperature under aggressivestirring (600 rpm). During the addition of chain extender solution,certain amount of solvent mixture (ethyl acetate/isopropanol) was addedinto the reaction mixture to maintain the reaction mixture at a suitableviscosity. After adding all the prepolymer solution into the jar, thereaction mixture was kept stirring for additional 5 h at roomtemperature to complete the chain extension. Similar procedures can befollowed in order to provide other biointerface polymers, as detailedherein.

Example 7 Synthesis of Enzyme Layer Polymer and Characterization Thereof

A betaine containing polyurethaneurea polymer was synthesized via atwo-step polycondensation reaction in organic solvents. In the firststep, a homogeneous polyurethane prepolymer with isocyanate end groupson both chain ends was prepared. In the second step, small moleculardiamine(s) was/were used as chain extender(s). These diamines react withprepolymer in dilute organic solution to obtain well-definedpolyurethaneurea with linear structure and narrow molecular weightdistribution.

As a representative example, the prepolymer was prepared by addingisophorone diisocyanate (IPDI), polyethylene oxide diol, polycarbonatediol, 2,2-bis(hydroxymethyl)propionic acid (Bis-MPA), and sulfobetaineprepolymer into a dry 200 mL reaction jar fitted with a nitrogen spargetube and a mechanical stirrer at room temperature. The reaction washeated to 65° C. for 30 min under nitrogen with mechanical stirring (200rpm) until all the reactants dissolved. 400 ppm of catalyst was addedinto the reaction, which was kept at 65° C. for 1 h. The reactiontemperature was raised to 85° C. and kept for 3 h until no bubbles wereobserved in the reaction mixture. The reaction mixture was allowed tostir for an additional 2 h at 100° C. to complete the formation of theprepolymer. The viscous prepolymer was cooled to 50° C. and dissolved inethyl acetate to form a transparent solution.

For the chain extension step, isophorone diamines were used as chainextenders and were added to a dry 700 mL reaction jar equipped with amechanical stirrer and diluted with ethyl acetate/isopropanol solventmixture. The polyurethane prepolymer solution was added dropwise intothe chain extender solution at room temperature under aggressivestirring. During the addition of chain extender solution, certain amountof solvent mixture (ethyl acetate/isopropanol) was added into thereaction mixture to maintain the reaction mixture at a suitableviscosity. After adding all the prepolymer solution into the reactionmixture, the reaction was kept stirring for additional 5 h at roomtemperature to complete the chain extension. The polymer thus formed wasdried in an oven at 50° C. under nitrogen flow to remove the solvent andre-dissolved or dispersed in a waterborne polyurethane dispersion alongwith enzyme and optionally crosslinking agent. The enzyme layer film wascast and dried at 50° C. for further characterization.

An active enzyme leaching assay measuring enzyme activity was used todetermine the amount of active enzyme leaching from a film. A film issoaked in solution and aliquots of the leachate at specific time pointsare measured for enzyme activity. Enzyme activity is determined by therate of hydrogen peroxide generated in the presence of excess glucose. Areactive dye in conjunction with peroxidase is quantitatively convertedby hydrogen peroxide to a colored compound and monitoredspectroscopically. The rate of colormetric change is related to theactivity of the sample, which reflects the active enzyme loading.Experiments are done on 200 μm thick films cast that are dried overnightin a 50° C. convection oven.

FIG. 34 illustrates % of enzyme leached from a control film (P3) and afilm prepared from the same polymer binder used in P3 but with 30 wt %of betaine-containing polymer as enzyme immobilization polymer additive,baseline subtracted. The results indicate effective GOX enzymeimmobilization with a betaine containing polymer.

Sensors were evaluated on calcheck metrics including sensitivity,baseline signal, oxygen sensitivity, linearity, and acetaminophenblocking. FIG. 35 shows certain sensor metrics (e.g. MARD and glucoseslope) for sensors having an enzyme layer formed of the same polymerbinder used in P3 but with 30 wt % of betaine containing polymer asenzyme immobilization polymer additive. Their performance under thevarious metrics are comparable. In this cal-check test, the followingcharacteristics were measured:

-   -   i. Glucose Slope (pA/mg/dL)—Ordinary least-squares linear        regression analysis of the electrical response of the sensor        when placed in buffer solutions of increasing glucose        concentration. It is also referred to as glucose sensitivity.    -   ii. Baseline Equivalent (mg/dL)—mg/dL equivalent of the        non-glucose related signal.    -   iii. MARD (%)—Mean Absolute Relative Difference, the measure of        variation away from the ideal line.    -   iv. Low Oxygen Response—Defined as the percent change in        electrical response under reduced oxygen conditions (i.e., at        0.25±0.05 mg O₂/L) compared with signal obtained under        atmospheric conditions. It is also referred to as Oxygen        Performance.    -   v. Acetaminophen Bias—the mg/dL equivalent signal from a 2 mg/dL        concentration of acetaminophen. Also referred to as glucose        equivalence.

TABLE 2 Enzyme layer polymer and characterization PEG Betaine HS Mn PDIName (wt. %) (wt. %) (wt. %) (Da) (Mw/Mn) Betaine-containing 55 7.6 2591,900 1.7 polymer additive WB-7 17 3.2 51 107,112 1.7 WB-8 17 3.2 5145,000 1.7 WB-9 16.6 3.2 50 N/A N/A WB-14 15.9 3.2 50 N/A N/A

Example 8 Synthesis of Enzyme Layer Polymer and Characterization thereof

A betaine containing polyurethaneurea polymer in aqueous solution wassynthesized via a two-step polycondensation reaction in water. In thefirst step, a homogeneous polyurethane prepolymer with isocyanate endgroups on both chain ends was prepared. In the second step, smallmolecular diamine(s) was/were used as chain extender(s). These diaminesreact with prepolymer in dilute aqueous solution to obtain waterbornepolyurethaneurea solution.

As a representative example, the prepolymer was prepared by addingpolyether diol, and 2,2-bis(hydroxymethyl)propionic acid into a dry 200mL reaction jar fitted with a nitrogen sparge tube and a mechanicalstirrer at room temperature. The reaction mixture was heated to 90° C.for 30 min under nitrogen with mechanical stirring (200 rpm) until allthe reactants melted and form transparent liquid. The reaction wasallowed to cool to 80° C. and added carboxybetaine diol, stir at 80° C.for 1 h. The reaction mixture was cooled down to 65° C. and then addedisophorone diisocyanate (IPDI). 400 ppm of catalyst was added into thereaction, and reaction was kept at 85° C. for 4 h under nitrogen withmechanical stirring. The reaction was neutralized with trimethylamineand then added into water dropwise to form prepolymer aqueous emulsion.

For the chain extension step, ethylenediamine were used as chainextenders and were added to a 700 mL reaction jar equipped with amechanical stirrer and diluted with water. The polyurethane prepolymeraqueous emulsion was added dropwise into the chain extender solution atroom temperature under aggressive stirring. After adding all theprepolymer solution into the reaction mixture, the reaction was keptstirring for additional 5 h at room temperature to complete the chainextension.

In a different assay as that detailed in Example 1, total enzymeleaching from enzyme layer films was determined using two separatetests. FIG. 34 uses a bicinchoninic acid test, which determines totalprotein content by peptide bond reducing of copper II ion to copper Iwith an associated color change of copper I complexing withbicinchoninic acid. This color change is measured via an absorptionmeasurement at 562 nm using a UV spectrophotometer.

Total eluted protein was also measured by a gel electrophoresis methodwith subsequent protein band quantification in FIG. 36. FIG. 36 showsthe comparison of enzyme leaching from film samples prepared fromstandard formulation (control, P3), standard formulation with theaddition of small molecule betaine additive (Ralufon), and film preparedfrom waterborne polyurethane dispersion with betaine incorporated intothe polymer as building-block, as disclosed in this example. Within 30minutes at room temperature, 200 μm thick samples of enzyme formulationcontrol (P3) leaches much more (2 orders of magnitudes) enzyme, thanwaterborne polyurethane dispersion betaine films, indicating that theenzyme is immobilized within the film. In addition, addition ofequivalent amount (3 wt %) of small molecule sulfo betaine Ralufon tothe P3 control formulation failed to improve the enzyme retention.Testing was continued to 24 hours, and waterborne polyurethanedispersion films continue to effectively retain enzyme.

Water adsorption of films made from enzyme layer solutions was performedat room temperature in water on 25-50 μm thick films. FIG. 37 indicatesthat betaine containing polymers as prepared in this example, WB-7 andstandard (P3) enzyme layer films both absorb most of the water they takeup within the first 5 minutes. The control films, however, almostimmediately start leaching a large amount of hydrophilic molecules,resulting in 10% loss of water, by weight, after 24 hours. Filmsprepared from betaine containing polyurethane dispersions with built-inbetaine have a stable hydrated state over time and are more hydrophilicand absorb more water than control enzyme formulation films.

Half sensors (sensors with all the components except a resistance layer)containing enzyme layer were subjected to high heat and humiditytreatment 70° C. and 95% humidity. A standard resistance layer was thenapplied after the treatment to avoid the effect of treatment onresistance layer and sensitivity was measured (FIG. 38). The data showthat higher sensitivity was maintained when using betaine containingpolymers in the enzyme layer.

The linearity was also determined and, as shown in FIG. 39, after thehumidity treatment the control (P3) enzyme sensors had poorer linearityand accuracy when compared to the sensors with betaine containingpolymers in the backbone.

Sensors containing enzyme layer WB-9 and WB-14 were prepared. A controlsensor (P3) was also used, which had a standard enzyme layer. After aperiod of soaking in heated buffer solution with and without glucose,membrane-coated sensors were transferred from a sensor fixture to aclamp fixture with a rubber pad. A silicone tube was transferred to theback end of the membrane-coated sensor using a syringe needle. Asilicone tube was placed on the syringe needle, the sensor was insertedinside the needle aperture (to protect membrane-coated area on sensor),and the silicone tube was then slide over the needle to reach back endof sensor. The silicone tube has a diameter small enough to be heldtightly in place on sensor. This was repeated for all sensors to betested. The clamp with sensors inserted in the silicone tube were soakedin same soaking buffer and temperature for 7 mins. Using a sensor dipperthe control P3 and carboxybetaine waterborne polymer sensors WB-9 andWB-14 were pulled at the same time with a fixed pulling speed, forcingthe sensor tip and skived region to go through silicone tube. This forceand speed motion is responsible of folding the membrane coating onsensors that do not have good layer adhesion. Sensors were examinedunder optical microscopy after the pulling test to identify the obviousmembrane delamination. The percent of sensors that passed theadhesion-pull test was determined by dividing the number of sensors thatfailed test (showed delamination) by the total amount of sensors thatwere tested, times 100. The results are shown in FIG. 40 and FIG. 41. Inboth cases the waterborne polymers WB-9 and WB-14 outperformed thestandard enzyme layers.

Example 9 Diffusion Resistance Layer

Sensors were prepared that contained a diffusion resistance layercomprising a blend of PVP and a polyurethane urea-polycarbonate blockcopolymer either with or without silicone. The block copolymer withsilicone had a T_(g) as shown in FIG. 43. The tensile strength of twoblock copolymers are shown in FIG. 44A, and the puncture resistance isshown in FIG. 44B. The sensitivity of the sensors were measured in an invitro model over 200 hours and the results are shown in FIG. 42. Thesensors with the high T_(g) and high tensile strength, silicone freepolymer in the diffusion resistance layer had minimal drift (less than10%) over the course of the experiment, as compared to the sensors withlower Tg and tensile strength and silicone.

Further the sensor noise was determined and the data is shown in FIGS.47A and 47B. The sensors with the high T_(g) and high tensile strength,silicone free polymer in the diffusion resistance layer exhibited lessin vivo noise in the 14 day study.

Example 10

Sensors were prepared that contained a diffusion resistance layercomprising a blend of PVP and a polyurethane urea-polycarbonate blockcopolymer either with or without silicone. The sensitivity of thesensors were tested in an in vivo pig model. At day 7 and day 14 thesensors were removed and analyzed to determine the percent of breach.Data is shown in FIG. 45. In terms of resistance layer mechanicalproperties on sensor, in vivo stability test indicate the mechanicalrobust high T_(g) polymer-based resistance layer has less breach rates.

Example 11

A sensor was prepared with a diffusion resistance layer comprising ablend of PVP and a polyurethane urea-polycarbonate block copolymerwithout silicone. In one sample, the sensor was soaked in distilledwater at 50° C. for various time intervals and then cured. In anothersample, the sensor was cured without soaking. The results are shown inFIG. 48. The soak cure eliminated drift from calcheck. FIG. 48 is acomparison of sensor sensitivity drift between a control sensor preparedfrom a silicone containing diffusion resistance layer and a test sensormade with a high T_(g) diffusion resistance layer, a polycarbonateurethane −1 as shown in FIG. 43. Sensors were exposed to a buffersolution with constant glucose concentration (300 mg/dL) and theirsensitivity were measured after subtracting background signal andmonitored over time up to 14 days. The sensitivity change over the 2 hrtime point were calculated and plotted in FIG. 48. As can be seen, thesensor prepared from a high T_(g) diffusion resistance layer experiencedmuch lower magnitude of drift compared to the one prepared from siliconecontaining diffusion resistance layer.

Methods and devices that are suitable for use in conjunction withaspects of the preferred embodiments are disclosed in U.S. Pat. Nos.4,757,022; 4,994,167; 6,001,067; 6,558,321; 6,702,857; 6,741,877;6,862,465; 6,931,327; 7,074,307; 7,081,195; 7,108,778; 7,110,803;7,134,999; 7,136,689; 7,192,450; 7,226,978; 7,276,029; 7,310,544;7,364,592; 7,366,556; 7,379,765; 7,424,318; 7,460,898; 7,467,003;7,471,972; 7,494,465; 7,497,827; 7,519,408; 7,583,990; 7,591,801;7,599,726; 7,613,491; 7,615,007; 7,632,228; 7,637,868; 7,640,048;7,651,596; 7,654,956; 7,657,297; 7,711,402; 7,713,574; 7,715,893;7,761,130; 7,771,352; 7,774,145; 7,775,975; 7,778,680; 7,783,333;7,792,562; 7,797,028; 7,826,981; 7,828,728; 7,831,287; 7,835,777;7,857,760; 7,860,545; 7,875,293; 7,881,763; 7,885,697; 7,896,809;7,899,511; 7,901,354; 7,905,833; 7,914,450; 7,917,186; 7,920,906;7,925,321; 7,927,274; 7,933,639; 7,935,057; 7,946,984; 7,949,381;7,955,261; 7,959,569; 7,970,448; 7,974,672; 7,976,492; 7,979,104;7,986,986; 7,998,071; 8,000,901; 8,005,524; 8,005,525; 8,010,174;8,027,708; 8,050,731; 8,052,601; 8,053,018; 8,060,173; 8,060,174;8,064,977; 8,073,519; 8,073,520; 8,118,877; 8,128,562; 8,133,178;8,150,488; 8,155,723; 8,160,669; 8,160,671; 8,167,801; 8,170,803;8,195,265; 8,206,297; 8,216,139; 8,229,534; 8,229,535; 8,229,536;8,231,531; 8,233,958; 8,233,959; 8,249,684; 8,251,906; 8,255,030;8,255,032; 8,255,033; 8,257,259; 8,260,393; 8,265,725; 8,275,437;8,275,438; 8,277,713; 8,280,475; 8,282,549; 8,282,550; 8,285,354;8,287,453; 8,290,559; 8,290,560; 8,290,561; 8,290,562; 8,292,810;8,298,142; 8,311,749; 8,313,434; 8,321,149; 8,332,008; 8,346,338;8,364,229; 8,369,919; 8,374,667; 8,386,004; and 8,394,021.

Methods and devices that are suitable for use in conjunction withaspects of the preferred embodiments are disclosed in U.S. PatentPublication No. 2003-0032874-A1; U.S. Patent Publication No.2005-0176136-A1; U.S. Patent Publication No. 2005-0182451-A1; U.S.Patent Publication No. 2005-0245799-A1; U.S. Patent Publication No.2005-0033132-A1; U.S. Patent Publication No. 2005-0051427-A1; U.S.Patent Publication No. 2005-0056552-A1; U.S. Patent Publication No.2005-0090607-A1; U.S. Patent Publication No. 2006-0015020-A1; U.S.Patent Publication No. 2006-0016700-A1; U.S. Patent Publication No.2006-0020188-A1; U.S. Patent Publication No. 2006-0020190-A1; U.S.Patent Publication No. 2006-0020191-A1; U.S. Patent Publication No.2006-0020192-A1; U.S. Patent Publication No. 2006-0036140-A1; U.S.Patent Publication No. 2006-0036143-A1; U.S. Patent Publication No.2006-0040402-A1; U.S. Patent Publication No. 2006-0068208-A1; U.S.Patent Publication No. 2006-0142651-A1; U.S. Patent Publication No.2006-0155180-A1; U.S. Patent Publication No. 2006-0198864-A1; U.S.Patent Publication No. 2006-0200020-A1; U.S. Patent Publication No.2006-0200022-A1; U.S. Patent Publication No. 2006-0200970-A1; U.S.Patent Publication No. 2006-0204536-A1; U.S. Patent Publication No.2006-0224108-A1; U.S. Patent Publication No. 2006-0235285-A1; U.S.Patent Publication No. 2006-0249381-A1; U.S. Patent Publication No.2006-0252027-A1; U.S. Patent Publication No. 2006-0253012-A1; U.S.Patent Publication No. 2006-0257995-A1; U.S. Patent Publication No.2006-0258761-A1; U.S. Patent Publication No. 2006-0263763-A1; U.S.Patent Publication No. 2006-0270922-A1; U.S. Patent Publication No.2006-0270923-A1; U.S. Patent Publication No. 2007-0027370-A1; U.S.Patent Publication No. 2007-0032706-A1; U.S. Patent Publication No.2007-0032718-A1; U.S. Patent Publication No. 2007-0045902-A1; U.S.Patent Publication No. 2007-0059196-A1; U.S. Patent Publication No.2007-0066873-A1; U.S. Patent Publication No. 2007-0173709-A1; U.S.Patent Publication No. 2007-0173710-A1; U.S. Patent Publication No.2007-0208245-A1; U.S. Patent Publication No. 2007-0208246-A1; U.S.Patent Publication No. 2007-0232879-A1; U.S. Patent Publication No.2008-0045824-A1; U.S. Patent Publication No. 2008-0083617-A1; U.S.Patent Publication No. 2008-0086044-A1; U.S. Patent Publication No.2008-0108942-A1; U.S. Patent Publication No. 2008-0119703-A1; U.S.Patent Publication No. 2008-0119704-A1; U.S. Patent Publication No.2008-0119706-A1; U.S. Patent Publication No. 2008-0183061-A1; U.S.Patent Publication No. 2008-0183399-A1; U.S. Patent Publication No.2008-0188731-A1; U.S. Patent Publication No. 2008-0189051-A1; U.S.Patent Publication No. 2008-0194938-A1; U.S. Patent Publication No.2008-0197024-A1; U.S. Patent Publication No. 2008-0200788-A1; U.S.Patent Publication No. 2008-0200789-A1; U.S. Patent Publication No.2008-0200791-A1; U.S. Patent Publication No. 2008-0214915-A1; U.S.Patent Publication No. 2008-0228054-A1; U.S. Patent Publication No.2008-0242961-A1; U.S. Patent Publication No. 2008-0262469-A1; U.S.Patent Publication No. 2008-0275313-A1; U.S. Patent Publication No.2008-0287765-A1; U.S. Patent Publication No. 2008-0306368-A1; U.S.Patent Publication No. 2008-0306434-A1; U.S. Patent Publication No.2008-0306435-A1; U.S. Patent Publication No. 2008-0306444-A1; U.S.Patent Publication No. 2009-0018424-A1; U.S. Patent Publication No.2009-0030294-A1; U.S. Patent Publication No. 2009-0036758-A1; U.S.Patent Publication No. 2009-0036763-A1; U.S. Patent Publication No.2009-0043181-A1; U.S. Patent Publication No. 2009-0043182-A1; U.S.Patent Publication No. 2009-0043525-A1; U.S. Patent Publication No.2009-0045055-A1; U.S. Patent Publication No. 2009-0062633-A1; U.S.Patent Publication No. 2009-0062635-A1; U.S. Patent Publication No.2009-0076360-A1; U.S. Patent Publication No. 2009-0099436-A1; U.S.Patent Publication No. 2009-0124877-A1; U.S. Patent Publication No.2009-0124879-A1; U.S. Patent Publication No. 2009-0124964-A1; U.S.Patent Publication No. 2009-0131769-A1; U.S. Patent Publication No.2009-0131777-A1; U.S. Patent Publication No. 2009-0137886-A1; U.S.Patent Publication No. 2009-0137887-A1; U.S. Patent Publication No.2009-0143659-A1; U.S. Patent Publication No. 2009-0143660-A1; U.S.Patent Publication No. 2009-0156919-A1; U.S. Patent Publication No.2009-0163790-A1; U.S. Patent Publication No. 2009-0178459-A1; U.S.Patent Publication No. 2009-0192366-A1; U.S. Patent Publication No.2009-0192380-A1; U.S. Patent Publication No. 2009-0192722-A1; U.S.Patent Publication No. 2009-0192724-A1; U.S. Patent Publication No.2009-0192751-A1; U.S. Patent Publication No. 2009-0203981-A1; U.S.Patent Publication No. 2009-0216103-A1; U.S. Patent Publication No.2009-0240120-A1; U.S. Patent Publication No. 2009-0240193-A1; U.S.Patent Publication No. 2009-0242399-A1; U.S. Patent Publication No.2009-0242425-A1; U.S. Patent Publication No. 2009-0247855-A1; U.S.Patent Publication No. 2009-0247856-A1; U.S. Patent Publication No.2009-0287074-A1; U.S. Patent Publication No. 2009-0299155-A1; U.S.Patent Publication No. 2009-0299156-A1; U.S. Patent Publication No.2009-0299162-A1; U.S. Patent Publication No. 2010-0010331-A1; U.S.Patent Publication No. 2010-0010332-A1; U.S. Patent Publication No.2010-0016687-A1; U.S. Patent Publication No. 2010-0016698-A1; U.S.Patent Publication No. 2010-0030484-A1; U.S. Patent Publication No.2010-0331644 A1; U.S. Patent Publication No. 2010-0036215-A1; U.S.Patent Publication No. 2010-0036225-A1; U.S. Patent Publication No.2010-0041971-A1; U.S. Patent Publication No. 2010-0045465-A1; U.S.Patent Publication No. 2010-0049024-A1; U.S. Patent Publication No.2010-0076283-A1; U.S. Patent Publication No. 2010-0081908-A1; U.S.Patent Publication No. 2010-0081910-A1; U.S. Patent Publication No.2010-0087724-A1; U.S. Patent Publication No. 2010-0096259-A1; U.S.Patent Publication No. 2010-0121169-A1; U.S. Patent Publication No.2010-0161269-A1; U.S. Patent Publication No. 2010-0168540-A1; U.S.Patent Publication No. 2010-0168541-A1; U.S. Patent Publication No.2010-0168542-A1; U.S. Patent Publication No. 2010-0168543-A1; U.S.Patent Publication No. 2010-0168544-A1; U.S. Patent Publication No.2010-0168545-A1; U.S. Patent Publication No. 2010-0168546-A1; U.S.Patent Publication No. 2010-0168657-A1; U.S. Patent Publication No.2010-0174157-A1; U.S. Patent Publication No. 2010-0174158-A1; U.S.Patent Publication No. 2010-0174163-A1; U.S. Patent Publication No.2010-0174164-A1; U.S. Patent Publication No. 2010-0174165-A1; U.S.Patent Publication No. 2010-0174166-A1; U.S. Patent Publication No.2010-0174167-A1; U.S. Patent Publication No. 2010-0179401-A1; U.S.Patent Publication No. 2010-0179402-A1; U.S. Patent Publication No.2010-0179404-A1; U.S. Patent Publication No. 2010-0179408-A1; U.S.Patent Publication No. 2010-0179409-A1; U.S. Patent Publication No.2010-0185065-A1; U.S. Patent Publication No. 2010-0185069-A1; U.S.Patent Publication No. 2010-0185070-A1; U.S. Patent Publication No.2010-0185071-A1; U.S. Patent Publication No. 2010-0185075-A1; U.S.Patent Publication No. 2010-0191082-A1; U.S. Patent Publication No.2010-0198035-A1; U.S. Patent Publication No. 2010-0198036-A1; U.S.Patent Publication No. 2010-0212583-A1; U.S. Patent Publication No.2010-0217557-A1; U.S. Patent Publication No. 2010-0223013-A1; U.S.Patent Publication No. 2010-0223022-A1; U.S. Patent Publication No.2010-0223023-A1; U.S. Patent Publication No. 2010-0228109-A1; U.S.Patent Publication No. 2010-0228497-A1; U.S. Patent Publication No.2010-0240975-A1; U.S. Patent Publication No. 2010-0240976 C1; U.S.Patent Publication No. 2010-0261987-A1; U.S. Patent Publication No.2010-0274107-A1; U.S. Patent Publication No. 2010-0280341-A1; U.S.Patent Publication No. 2010-0286496-A1; U.S. Patent Publication No.2010-0298684-A1; U.S. Patent Publication No. 2010-0324403-A1; U.S.Patent Publication No. 2010-0331656-A1; U.S. Patent Publication No.2010-0331657-A1; U.S. Patent Publication No. 2011-0004085-A1; U.S.Patent Publication No. 2011-0009727-A1; U.S. Patent Publication No.2011-0024043-A1; U.S. Patent Publication No. 2011-0024307-A1; U.S.Patent Publication No. 2011-0027127-A1; U.S. Patent Publication No.2011-0027453-A1; U.S. Patent Publication No. 2011-0027458-A1; U.S.Patent Publication No. 2011-0028815-A1; U.S. Patent Publication No.2011-0028816-A1; U.S. Patent Publication No. 2011-0046467-A1; U.S.Patent Publication No. 2011-0077490-A1; U.S. Patent Publication No.2011-0118579-A1; U.S. Patent Publication No. 2011-0124992-A1; U.S.Patent Publication No. 2011-0125410-A1; U.S. Patent Publication No.2011-0130970-A1; U.S. Patent Publication No. 2011-0130971-A1; U.S.Patent Publication No. 2011-0130998-A1; U.S. Patent Publication No.2011-0144465-A1; U.S. Patent Publication No. 2011-0178378-A1; U.S.Patent Publication No. 2011-0190614-A1; U.S. Patent Publication No.2011-0201910-A1; U.S. Patent Publication No. 2011-0201911-A1; U.S.Patent Publication No. 2011-0218414-A1; U.S. Patent Publication No.2011-0231140-A1; U.S. Patent Publication No. 2011-0231141-A1; U.S.Patent Publication No. 2011-0231142-A1; U.S. Patent Publication No.2011-0253533-A1; U.S. Patent Publication No. 2011-0263958-A1; U.S.Patent Publication No. 2011-0270062-A1; U.S. Patent Publication No.2011-0270158-A1; U.S. Patent Publication No. 2011-0275919-A1; U.S.Patent Publication No. 2011-0290645-A1; U.S. Patent Publication No.2011-0313543-A1; U.S. Patent Publication No. 2011-0320130-A1; U.S.Patent Publication No. 2012-0035445-A1; U.S. Patent Publication No.2012-0040101-A1; U.S. Patent Publication No. 2012-0046534-A1; U.S.Patent Publication No. 2012-0078071-A1; U.S. Patent Publication No.2012-0108934-A1; U.S. Patent Publication No. 2012-0130214-A1; U.S.Patent Publication No. 2012-0172691-A1; U.S. Patent Publication No.2012-0179014-A1; U.S. Patent Publication No. 2012-0186581-A1; U.S.Patent Publication No. 2012-0190953-A1; U.S. Patent Publication No.2012-0191063-A1; U.S. Patent Publication No. 2012-0203467-A1; U.S.Patent Publication No. 2012-0209098-A1; U.S. Patent Publication No.2012-0215086-A1; U.S. Patent Publication No. 2012-0215087-A1; U.S.Patent Publication No. 2012-0215201-A1; U.S. Patent Publication No.2012-0215461-A1; U.S. Patent Publication No. 2012-0215462-A1; U.S.Patent Publication No. 2012-0215496-A1; U.S. Patent Publication No.2012-0220979-A1; U.S. Patent Publication No. 2012-0226121-A1; U.S.Patent Publication No. 2012-0228134-A1; U.S. Patent Publication No.2012-0238852-A1; U.S. Patent Publication No. 2012-0245448-A1; U.S.Patent Publication No. 2012-0245855-A1; U.S. Patent Publication No.2012-0255875-A1; U.S. Patent Publication No. 2012-0258748-A1; U.S.Patent Publication No. 2012-0259191-A1; U.S. Patent Publication No.2012-0260323-A1; U.S. Patent Publication No. 2012-0262298-A1; U.S.Patent Publication No. 2012-0265035-A1; U.S. Patent Publication No.2012-0265036-A1; U.S. Patent Publication No. 2012-0265037-A1; U.S.Patent Publication No. 2012-0277562-A1; U.S. Patent Publication No.2012-0277566-A1; U.S. Patent Publication No. 2012-0283541-A1; U.S.Patent Publication No. 2012-0283543-A1; U.S. Patent Publication No.2012-0296311-A1; U.S. Patent Publication No. 2012-0302854-A1; U.S.Patent Publication No. 2012-0302855-A1; U.S. Patent Publication No.2012-0323100-A1; U.S. Patent Publication No. 2013-0012798-A1; U.S.Patent Publication No. 2013-0030273-A1; U.S. Patent Publication No.2013-0035575-A1; U.S. Patent Publication No. 2013-0035865-A1; U.S.Patent Publication No. 2013-0035871-A1; U.S. Patent Publication No.2013-0053665-A1; U.S. Patent Publication No. 2013-0053666-A1; US. PatentPublication No. 2013-0060112-A1; US. Patent Publication No.2013-0078912-A1; US. Patent Publication No. 2013-0076531-A1; US. PatentPublication No. 2013-0076532-A1; US. Patent Publication No.2013-0131478-A1; US. Patent Publication No. 2013-150692-A1; U.S. PatentPublication No. 2014-0094671-A1; US. Patent Publication No.2014-0005508-A1; US. Patent Publication No. 2014-0118166-A1; US. PatentPublication No. 2014-0118138-A1; US. Patent Publication No.2014-0188402-A1; US. Patent Publication No. 2014-0182350-A1; and US.Patent Publication No. 2014-0275896-A1.

Methods and devices that are suitable for use in conjunction withaspects of the preferred embodiments are disclosed in U.S. applicationSer. No. 09/447,227 filed on Nov. 22, 1999 and entitled “DEVICE ANDMETHOD FOR DETERMINING ANALYTE LEVELS,” and U.S. application Ser. No.13/461,625 filed on May 1, 2012 and entitled “DUAL ELECTRODE SYSTEM FORA CONTINUOUS ANALYTE SENSOR.”

For ease of explanation and illustration, in some instances the detaileddescription describes exemplary systems and methods in terms of acontinuous glucose monitoring environment; however it should beunderstood that the scope of the invention is not limited to thatparticular environment, and that one skilled in the art will appreciatethat the systems and methods described herein can be embodied in variousforms. Accordingly any structural and/or functional details disclosedherein are not to be interpreted as limiting the systems and methods,but rather are provided as attributes of a representative embodimentand/or arrangement for teaching one skilled in the art one or more waysto implement the systems and methods, which may be advantageous in othercontexts.

For example, and without limitation, described monitoring systems andmethods may include sensors that measure the concentration of one ormore analytes (for instance glucose, lactate, potassium, pH,cholesterol, isoprene, and/or hemoglobin) and/or other blood or bodilyfluid constituents of or relevant to a host and/or another party.

By way of example, and without limitation, monitoring system and methodembodiments described herein may include finger-stick blood sampling,blood analyte test strips, non-invasive sensors, wearable monitors (e.g.smart bracelets, smart watches, smart rings, smart necklaces orpendants, workout monitors, fitness monitors, health and/or medicalmonitors, clip-on monitors, and the like), adhesive sensors, smarttextiles and/or clothing incorporating sensors, shoe inserts and/orinsoles that include sensors, transdermal (i.e. transcutaneous) sensors,and/or swallowed, inhaled or implantable sensors.

In some embodiments, and without limitation, monitoring systems andmethods may comprise other sensors instead of or in additional to thesensors described herein, such as inertial measurement units includingaccelerometers, gyroscopes, magnetometers and/or barometers; motion,altitude, position, and/or location sensors; biometric sensors; opticalsensors including for instance optical heart rate monitors,photoplethysmogram (PPG)/pulse oximeters, fluorescence monitors, andcameras; wearable electrodes; electrocardiogram (EKG or ECG),electroencephalography (EEG), and/or electromyography (EMG) sensors;chemical sensors; flexible sensors for instance for measuring stretch,displacement, pressure, weight, or impact; galvanometric sensors,capacitive sensors, electric field sensors, temperature/thermal sensors,microphones, vibration sensors, ultrasound sensors,piezoelectric/piezoresistive sensors, and/or transducers for measuringinformation of or relevant to a host and/or another party.

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Thedisclosure is not limited to the disclosed embodiments. Variations tothe disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed disclosure, from a study ofthe drawings, the disclosure and the appended claims.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof; adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

What is claimed is:
 1. A device for monitoring an analyte concentration,the device comprising: a transcutaneous sensor configured to generate asignal associated with a concentration of an analyte; an enzyme layer;and a membrane located over both the transcutaneous sensor and theenzyme layer; wherein the membrane comprises a diffusion-resistancelayer comprising a base polymer, the base polymer comprising silicone,wherein the silicone comprises less than 1 wt % of the base polymer, andthe base polymer has a lowest glass transition temperature as measuredusing ASTM D3418 of greater than −50° C., and the base polymer has anultimate tensile strength as measured by ASTM D1708 that is greater than6000 psi.
 2. The device of claim 1, wherein the lowest glass transitiontemperature of the base polymer is greater than 0° C.
 3. The device ofclaim 1, wherein the lowest glass transition temperature of the basepolymer is from 0° C. to 66° C.
 4. The device of claim 1, wherein thelowest glass transition temperature of the base polymer is from 20° C.to 60° C.
 5. The device of claim 1, wherein the lowest glass transitiontemperature of the base polymer is from 0° C. to 30° C.
 6. The device ofclaim 1, wherein the lowest glass transition temperature of the basepolymer is from 30° C. to 60° C.
 7. The device of claim 1, wherein thebase polymer has an ultimate tensile strength greater than 8250 psi. 8.The device of claim 1, wherein the base polymer is a segmented blockcopolymer.
 9. The device of claim 1, wherein the base polymer comprisespolyurethane and/or polyurea segments and one or more polycarbonate orpolyester segments.
 10. The device of claim 1, wherein the base polymeris a polyurethane copolymer chosen from a polycarbonate-urethane,polyether-urethane, and polyester-urethane.
 11. The device of claim 1,wherein the base polymer comprises a polymer selected from the groupconsisting of epoxies, polystyrene, polyoxymethylene, polysiloxanes,polyethers, polyacrylics, polymethacrylic, polyesters, polycarbonates,polyamide, poly(ether ketone), and poly(ether imide).
 12. The device ofclaim 1, wherein the diffusion-resistance layer further comprises ahydrophilic polymer.
 13. The device of claim 12, wherein the hydrophilicpolymer is selected from the group consisting of polyvinyl alcohol,polyethylene glycol, polyacrylamide, polyacetate, polyethylene oxide,polyethyleneamine, polyvinylpyrrolidone, polyoxazoline, and mixturesthereof.
 14. The device of claim 12, wherein the hydrophilic polymer isblended with the base polymer.
 15. The device of claim 12, wherein thehydrophilic polymer is covalently bonded to the base polymer.
 16. Thedevice of claim 12, wherein the base polymer or hydrophilic polymercomprises at least one crosslinker, wherein the at least one crosslinkercomprises a polymer or an oligomer, the oligomer comprisingpolyfunctional isocyanate, polyfunctional aziridine, or polyfunctionalcarbodiimide.
 17. The device of claim 1, wherein thediffusion-resistance layer comprises a blend of a polycarbonate-urethanebase polymer and polyvinylpyrrolidone.
 18. The device of claim 1,wherein the diffusion-resistance layer is from 0.01 μm to about 250 μmthick.
 19. The device of claim 1, wherein the sensor has a drift of lessthan or equal to 10% over 10 days.
 20. The device of claim 1, whereinthe transcutaneous sensor comprises an electrode.
 21. The device ofclaim 1, wherein the device is configured for continuous measurement ofan analyte concentration.
 22. The device of claim 1, wherein the analyteis glucose.
 23. The device of claim 1, wherein the base polymer has aplurality of glass transition temperatures as measured using ASTM D3418.